Lipoxin Analogs as Novel Inhibitors of Angiogenesis

ABSTRACT

The present invention is directed to methods for the prevention or inhibition of angiogenesis. The method is accomplished by the administration of an effective amount of 15-epi-16-(para-fluoro)-phenoxy-lipoxin A 4 , LXA 4 , 15-epi-LXA 4  or 15-R/S-methyl, LXA 4  and pharmaceutically acceptable salts, esters, amides, carboxylic acids, or prodrugs thereof, to a subject in need thereof. As a consequence of the action of the therapeutic agent, angiogenesis is prevented or inhibited in the subject.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a Continuation of U.S. patent application Ser. No.11/222,458, filed Sep. 8, 2005, which is a Divisional of U.S. patentapplication Ser. No. 10/615,361, filed Jul. 8, 2003, which id aDivisional of U.S. patent application Ser. No. 10/086,609, filed Mar. 1,2002, now U.S. Pat. No. 6,627,658, which claims benefit to U.S.Provisional Application No. 60/272,931, filed Mar. 2, 2001, the contentsof which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. government may have rights in this invention pursuant toNational Institute of Health Grant Nos. GM38765 and P01-DE13499.

BACKGROUND OF THE INVENTION

Angiogenesis is a fundamental process by which new capillaries areformed from existing blood vessels. This process plays important rolesin physiological events such as formation of the corpus luteum,development of the embryo and wound healing, including recovery fromboth myocardial ischemia and peptic ulcer (1). Unregulated growth ofblood vessels can contribute to tissue injury in a large number ofdiseases such as arthritis, diabetes, and tumor progression (2).Endothelial cells are normally quiescent and are activated during theangiogenic response. Upon stimulation, endothelial cells can degradetheir basement membrane and proximal extracellular matrix, migratedirectionally, then divide and organize into functional capillariesinvested by a new basal lamina (3).

There is a growing body of evidence demonstrating that the angiogenicswitch is regulated by the net balance between positive and negativeregulators of new capillary growth (2). Persistence ofneovascularization requires a pro-angiogenic environment, with theexpression of angiogenic factors outweighing that of angiostaticfactors. A range of peptides can influence this balance, includingmitogenic factors such vascular endothelial growth factor (VEGF) (3),nonmitogenic factors (selected cytokines, CXC chemokines), and internalpeptide fragments of angiostatin and endostatin (3). Certain eicosanoidsalso have potent biologic actions on vascular endothelial cells. Inrabbits, PGE₂, PGR_(2α), and prostacylin (PGI₂) stimulate angiogenesiswhere prostaglandin E series, in particular PGE₁, is most potent. PGE₂is a potent inducer of VEGF expression in synovial fibroblasts. Inaddition to its known vasodilator and antiplatelet properties, PGI₂ canalso induce VEGF gene expression and protein synthesis (4).

It was recently reported that 12-lipoxygenase activity and one of itsproducts, 12(S)-HETE, is required for angiogenic responses (5), and thatP450-derived 12R-HETE stimulates angiogenesis via NF-kB (6). Thecyclooxygenase-2 (COX-2) gene in endothelial cells is rapidlyupregulated by several growth factors as well as inducers ofangiogenesis (7). Along these lines, results using three differentendothelial cell models show that COX-2 is an essential component ofangiogenesis, at least in vitro (8). Nonsteroidal anti-inflammatorydrugs such as aspirin (ASA) have been implicated in the prevention ofcertain cancers such as lung and colon cancer (9, 10) that might berelated to ASA's ability to reduce angiogenesis (7).

A need therefore exists, for compositions and methods to preventangiogenesis that are directed toward the disease process, such thatangiogenesis is prevented or inhibited physiologically. A need alsoexists for compositions and methods that induce angiogenesis in tissuethat is lacking the requisite or essential physiological requirementsfor sustainability.

SUMMARY

Proliferative states such as chronic inflammation, ischemic diseases andcancer are often accompanied by intense angiogenesis, a highlyorchestrated process involving vessel sprouting, endothelial cellmigration, proliferation and maturation. Aspirin-triggered lipoxins(ATL), the 15R enantiomeric counterparts of lipoxins (LXs), areendogenous mediators generated during multicellular responses thatdisplay potent immunomodulatory actions. Surprisingly, it has beendiscovered that LXs, ATLs, and more specifically, the ATL stableanalogs, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ (denoted ATL-1),LXA_(4,) 15-epi-LXA₄ and 15-R/S-methyl, LXA₄ are potent inhibitors ofangiogenesis. For example, ATL-1, LXA₄, 15-epi-LXA₄ and 15-R/S-methyl,LXA₄ each inhibited endothelial cell proliferation in the 1-10 nM rangeby 50% in cells stimulated with either vascular endothelial growthfactor (VEGF) at 3 ng/ml or leukotriene D₄ (10 nM). In addition, ATL-1(in a 10-100 nM range) inhibited VEGF (3 ng/ml)—induced endothelial cellchemotaxis. In a granuloma in vivo model of inflammatory angiogenesis,ATL-1 treatment (10 μg/mouse) reduced by ˜50% the angiogenic phenotype,as assessed by both vascular casting and fluorescence. Together, theseresults identified a novel and potent previously unappreciated action ofaspirin-triggered 15-epi-LX.

In one aspect, the present invention pertains to methods for theprevention, reduction or inhibition of angiogenesis. The method isaccomplished by the administration of an effective amount of LXA₄ andanalogs thereof, such as 15-R/S methyl, LXA₄, and pharmaceuticallyacceptable salts, esters, amides or prodrugs thereof, to a subject inneed thereof. As a consequence of the action of the therapeutic agent,angiogenesis is prevented or inhibited in the subject.

In another aspect, the present invention also pertains to methods forthe prevention or inhibition of angiogenesis. The method is accomplishedby the administration of an effective amount of an aspirin triggeredlipoxin (ATL) (15-epi-LXA₄, such as15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ (ATL-1)), andpharmaceutically acceptable salts, esters, amides or prodrugs thereof,to a subject in need thereof. As a consequence of the action of thetherapeutic agent, angiogenesis is prevented or inhibited in thesubject.

In still another aspect, the present invention pertains to methods forthe prevention or inhibition of solid tumor tissue growth undergoingneovascularization in a subject. The method is accomplished by theadministration of an effective amount of an aspirin triggered lipoxin(ATL) (15-epi-LXA₄, such as 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄(ATL-1)), and pharmaceutically acceptable salts, esters, amides orprodrugs thereof, to a subject in need thereof, to a subject in needthereof.

In another aspect, the present invention pertains to methods for theprevention or inhibition of solid tumor tissue growth undergoingneovascularization in a subject. The method is accomplished by theadministration of an effective amount of LXA₄ and analogs thereof, suchas 15-R/S methyl, LXA₄, and pharmaceutically acceptable salts, esters,amides or prodrugs thereof, to a subject in need thereof.

In yet another aspect, the present invention is directed to methods toinhibit or prevent neovascularization from occurring in a subject. Themethod is accomplished by the administration of an effective amount ofLXA₄ and analogs thereof, such as 15-R/S methyl, LXA₄, andpharmaceutically acceptable salts, esters, amides or prodrugs thereof,to a subject in need thereof.

In yet another aspect, the present invention is directed to methods toinhibit or prevent neovascularization from occurring in a subject. Themethod is accomplished by the administration of an effective amount ofan aspirin triggered lipoxin (ATL) (15-epi-LXA₄, such as15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ (ATL-1)), andpharmaceutically acceptable salts, esters, amides or prodrugs thereof,to a subject in need thereof.

The invention is also directed to methods for treating a subject inwhich neovascularization is occurring in retinal tissue. Theneovascularization in the retinal tissue can be prevented or inhibitedby administering an effective amount of LXA₄ and analogs thereof, suchas 15-R/S methyl, LXA₄, and pharmaceutically acceptable salts, esters,amides or prodrugs thereof, to a subject in need thereof.

The present invention is further directed to methods for treating asubject in which neovascularization is occurring in retinal tissue. Theneovascularization in the retinal tissue can be prevented or inhibitedby administering an effective amount of an aspirin triggered lipoxin(ATL) (15-epi-LXA₄, such as 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄(ATL-1)), and pharmaceutically acceptable salts, esters, amides orprodrugs thereof, to a subject in need thereof.

The invention is further directed to methods for treating a subject forrestenosis in tissue wherein smooth muscle cell migration occursfollowing angioplasty. The restenosis can be prevented or inhibited byadministering an effective amount of LXA₄ and analogs thereof, such as15-R/S methyl, LXA₄, and pharmaceutically acceptable salts, esters,amides or prodrugs thereof, to a subject in need thereof.

The invention is directed to methods for treating a subject forrestenosis in tissue wherein smooth muscle cell migration occursfollowing angioplasty. The restenosis can be prevented or inhibited byadministering an effective amount of an aspirin triggered lipoxin (ATL)(15-epi-LXA₄, such as 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄(ATL-1)), and pharmaceutically acceptable salts, esters, amides orprodrugs thereof, to a subject in need thereof.

In still another embodiment, the invention pertains to methods ofreducing blood supply to tissue required to support new growth of thetissue in a subject. This reduction or elimination of new undesiredgrowth of tissue can be accomplished by the administration of acomposition comprising an effective amount of LXA₄ and analogs thereof,such as 15-R/S methyl, LXA₄, and pharmaceutically acceptable salts,esters, amides or prodrugs thereof, to a subject in need thereof.

The invention further pertains to methods of reducing blood supply totissue required to support new growth of the tissue in a subject. Thisreduction or elimination of new undesired growth of tissue can beaccomplished by the administration of a composition comprising aneffective amount of an aspirin triggered lipoxin (ATL) (15-epi-LXA₄,such as 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ (ATL-1)), andpharmaceutically acceptable salts, esters, amides or prodrugs thereof,to a subject in need thereof.

Further, the present invention pertains to methods for the prevention,diminishment or inhibition the production of new vessels in a subjectassociated with or stimulated by the production or release of VEGF. Themethod is accomplished by the administration of an effective amount of atherapeutic agent, including LXA₄ and analogs thereof, such as 15-R/Smethyl, LXA₄, and pharmaceutically acceptable salts, esters, amides orprodrugs thereof, to a subject in need thereof. Alternatively, aneffective amount of an aspirin triggered lipoxin (ATL) (15-epi-LXA₄,such as 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ (ATL-1)), andpharmaceutically acceptable salts, esters, amides or prodrugs thereofcan be utilized. As a consequence of the action of the therapeuticagent, growth of new vessels associated with the production of VEGF isprevented or inhibited and thus the growth of endothelial cells in thesubject. For example, VEGF is associated with tumor genesis,lymphoangiogenesis and proliferative disorders. The invention therefore,can be used to inhibit, reduce or prevent metasasis of tumors.

Surprisingly, configurational isomers of LXA₄, LXA₄ analogs and ATLanalogs, LXB₄ and LXB₄ analogs and pharmaceutically acceptable salts,esters, amides or prodrugs thereof, provide the opposite effects withregard to revascularization of tissue by the above-identified compoundsof the invention. That is, it has been surprisingly discovered that LXB₄and LXB₄ analogs have the ability to stimulate regeneration and ingrowthof vascular or epithelial tissue in tissues that are in need of suchstimulation. This is especially important in tissue grafting, tissueengineering and prosthetic group sites of attachment.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 demonstrates that ATL-1 inhibits VEGF-stimulated HUVECproliferation. HUVEC (5×10³) were plated in 96-well culture plates andcell proliferation was stimulated with 3 ng/ml VEGF. Three days aftertreatment, cell numbers were measured using MTT assay. Results areexpressed as percent inhibition of proliferation relative to vehicle andrepresent mean±SE for four independent experiments performed intriplicate. Inset: Representative experiment showing the time course ofcell proliferation induced by 3 ng/ml (filled triangle) or 10 ng/ml(filled square) VEGF. Vehicle (open circles) and ATL-1 (100 nM) (opentriangle).

FIG. 2 demonstrates that ATL-1, as well as LXA₄, 15-epi-LXA₄ and15-R/S-methyl, LXA₄ each inhibit endothelial cell chemotaxis. (A)Chemotaxis was initiated by addition of VEGF (3 ng/ml) or ATL-1 (100 nM)to the lower compartment of a 48-well chemotaxis chamber. Results areexpressed as percent of cell migration compared to vehicle alone andrepresent mean±SE for three independent experiments performed intriplicate (P<0.05). (B) HUVEC were incubated with vehicle or indicatedconcentrations of ATL-1 (15 min, 37° C.) and added to the uppercompartment of the microchamber (1×10⁶/well). Chemotaxis was initiatedby addition of VEGF (3 ng/ml) to the lower compartment. Results areexpressed as percent inhibition of migration relative to VEGF for arepresentative experiment performed in triplicate.

FIG. 3 is a graphical representation demonstrating inhibition of HUVECproliferation. (A) ATL-1 inhibits LTD₄-stimulated HUVEC proliferation.HUVECs (5×10³) were plated in 96-well culture plates, cell proliferationwas stimulated with 10 nM LTD₄, and cell numbers were determined after 3days using MTT. (B), concentration dependent cell proliferation inducedby LTD₄ and LTB₄. Results are expressed as mean±S.E. for fourindependent experiments performed in triplicate.

FIG. 4 demonstrates that ATL-1 inhibits angiogenic phenotype in vivo.(A) Vascular index (VI=mg carmine dye/mg weight of tissue) in day-6murine air pouch. Animals received a local injection of ATL-1 (10 μg/pouch) or vehicle immediately before VEGF (1 μg/pouch), 24 h afterraising the pouch. Results are expressed as the mean±SE for n=4 animalsper group. *Denotes statistically significant difference (P<0.05) fromVEGF alone; (B) Cedar wood oil histology of air pouch. Carmine dyevascular casts were made in day-6 air pouch from mice treated locallywith vehicle, ATL-1 (10 μg), VEGF (1 μg) or VEGF plus ATL-1. Tissue wasfixed in ethanol and cleared in cedarwood oil.

FIG. 5 depicts Anti-angiogenic action of ATL-1: fluorescent microscopy.Representative fluorescence photomicrographs showing the anti-angiogenicaction of ATL-1 (10 μg/pouch) in the murine air pouch (see methods).

FIG. 6 are photomicrographs of a murine air pouch. Immunohistochemistryfor murine air pouch CD31. Paraffin-embedded air pouch sections werestained for CD31 from mice as in FIGS. 4 and 5 and treated with vehiclealone (A), analog alone (B), VEGF-treated mice (C), and VEGF plusATL-treated mice (D). Results are representative of eight separate miceeach in duplicate. Magnification is 200× power and horseradishperoxidase with hematoxylin counterstain.

DETAILED DESCRIPTION

The features and other details of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

Abbreviations used throughout the present application include thefollowing and are included here for convenience. ASA, aspirin; ATL,aspirin-triggered 15-epi-lipoxins; ATL-1,15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄; COX, cyclooxygenase; HETE,hydroxyeicosatetraenoic acid; HUVEC, human umbilical vein endothelialcells; IL, interleukin; LO, lipoxygenase; LT, leukotriene; LX, lipoxin;LXA_(4,) 5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid;15-epi-LXA₄, 5S,6R,15R-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoicacid; 15-R/S-methyl, LXA₄,5S,6R,15R/S-trihydroxy-15-methyl-7,9,13-trans-11-cis-eicosatetraenoicacid, methyl ester; LXB₄,5S,14R,15S-trihydroxy-6,8,12-trans-10-cis-eicosatetraenoic acid; MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PBS, phosphatebuffered saline; PG, prostaglandin; PMN, neutrophils; VEGF, vascularendothelial growth factor.

It is to be understood, that throughout the present specification,reference is often made to the therapeutic compounds of the inventionsas esters, for example, ATL-1 as a carboxylic ester, i.e., methyl ester.However, all pharmaceutically acceptable salts, esters, amides, andprodrugs, including the carboxylic acid, are considered within the scopeof the invention for the LXA₄, ATL and LXB₄ compounds. For convenience,this terminology has been minimized throughout the description butshould be considered as part of the invention. Additionally, it shouldbe understood that the terms LXA₄, 15-epi-LXA₄ and 15-R/S-methyl, LXA₄also include all pharmaceutically acceptable salts, esters, amides,prodrugs and carboxylic acids.

Additionally, the hydroxyl(s) of ATLs, LXA₄s, and LXB₄s can be protectedby various protecting groups, such as those known in the art. An artisanskilled in the art can readily determine which protecting group(s) canbe useful for the protection of the hydroxyl group(s). Standard methodsare known in the art and are more fully described in literature. Forexample, suitable protecting groups can be selected by the skilledartisan and are described in Green and Wuts, “Protecting Groups inOrganic Synthesis”, John Wiley and Sons, 1991, the teachings of whichare incorporated herein by reference. Preferred protecting groupsinclude TMS or TIPPS groups, and preferably acetate or proprionategroups.

For example, one or more hydroxyl groups can be treated with a mildbase, such as triethylamine in the presence of an acid chloride or silylchloride to facilitate a reaction between the hydroxyl ion and thehalide. Alternatively, an alkyl halide can be reacted with the hydroxylion (generated by a base such as lithium diisopropyl amide) tofacilitate ether formation.

It should also be understood that not all hydroxyl groups need beprotected. One, two or all three hydroxyl groups can be protected. Thiscan be accomplished by the stoichiometric choice of reagents used toprotect the hydroxyl groups. Methods known in the art can be used toseparate the mono, di- or tri-protected hydroxy compounds, e.g., HPLC,LC, flash chromatography, gel permeation chromatography,crystallization, distillation, etc.

One advantage of protecting one or more hydroxyl groups of ATL, LXA₄, orLXB₄ compounds, e.g., via acetates, is the ability to delay the completemetabolic uptake of the compound(s). This is one means by which thecompound(s) can remain active over a prolonged period of time as thesubject's body slowly removes the protecting group from the hydroxylunder normal physiological conditions. Additionally, by protecting oneor more of the hydroxyl groups of these compounds, hydrolysis of theprotecting group allows the medication to enter the biochemical pathwayof the subject prior to degradation of the parent, unprotected,compound.

Methods to prepare lipoxin analogs (ATLs, LXA₄s, or LXB₄s) are known inthe art. For example, U.S. Pat. Nos. 4,576,758, 4,560,514, 5,079,261,5,049,681, 5,441,951, 5,648,512, 5,650,435, 6,048,897, 6,100,296,6,177,468 and 6,316,648 and Japanese Patent Nos. 3,227,922, 63,088,153,62,198,677 and 1,228,994 describe approaches to prepare lipoxin analogs.Publications by K. C. Nicolaou et al. include approaches to variouslipoxin compounds. (For example see, Nicolaou, K. C. et al. Biochim.Biophys. Acta 1003:44-53; Nicolaou, K. C. et al. J. Org. Chem.54:5527-5535; and Nicolaou, K. C. Angew. Chem. Int. Ed. Engl.30:1100-1116. Additional literature references for the preparation oflipoxin analogs include Takano, T., S. Fiore, J. F. Maddox, H. R. Brady,N. A. Petasis, and C. N. Serhan. 1997. Aspirin-triggered 15-epi-lipoxinA₄ and LXA₄ stable analogs are potent inhibitors of acute inflammation:Evidence for anti-inflammatory receptors. J. Exp. Med. 185:1693-1704 andSerhan, Charles N., Maddox, Jane F., Petasis, Nicos A.,Akritopoulou-Zanze, Irini, Papayianni, Aikaterina, Brady, Hugh R.,Colgan, Sean P., and Madara, James L. (1995), Biochemistry, 34, pp.14609-14614.

Aspirin's therapeutic mechanism of action includes inhibition ofCOX-derived prostanoids (10). It was discovered that COX-2, whenacetylated by ASA, blocks the ability of COX-2 to generate prostanoids,yet this enzyme remains active in endothelial cells, epithelial cellsand mononuclear cells and initiates the biosynthesis of new products ofcell-cell interactions or transcellular biosynthesis termedaspirin-triggered-15-epi-lipoxins (ATLs) (11). These novel endogenouslipid mediators are the carbon 15 epimers of LX that carry their 15alcohol in the R configuration compared to their native lipoxin (LX)counterparts and appear to mimic most if not all endogenous LXbioactivities.

To date the actions of ATLs appear to be most relevant in regulatinginflammatory responses, since they are generated during cell-cellinteractions that can involve, for example, endothelialcells-neutrophils in vivo (12), and display potent inhibitory actions inseveral key and strategic events in inflammation (12-14). Both LX andATL actions include inhibiting adhesion and transmigration ofneutrophils, and hence can serve as counter regulatory signals to limitand/or regulate leukocyte accumulation that are potentially operative inthe dampening and resolution of inflammatory sites (14). Since LX arerapidly generated and inactivated in the local microenvironment, toinvestigate these actions in vivo, stable analogs of both lipoxins,i.e., LXA₄ and ATL were designed that enhance bioavailabilities andthese natural compounds bioactivities compared to their native products(14) and also proved to be ˜100 times the potency of ASA (13). Thepresent invention establishes that LXA₄s and ATLs can regulateangiogenesis, a previously unknown and surprising application of thesecompounds. For example, using a metabolically more stable ATL syntheticanalog [15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ (denoted ATL-1)],ATLs and LXA₄ compounds, including, 15-epi-LXA₄ or 15-R/S-methyl, LXA₄and LXA₄ proved to be potent angiostatic eicosanoid in vivo, identifyinga new activity for these endogenous mediators that is in sharp contrastto the actions of other eicosanoids and is relevant in several humandiseases.

In one aspect, the present invention pertains to methods for theprevention, diminishment or inhibition of angiogenesis. The method isaccomplished by the administration of an effective amount of LXA₄ andanalogs thereof, such as 15-R/S methyl, LXA₄, and pharmaceuticallyacceptable salts, esters, amides or prodrugs thereof, to a subject inneed thereof. As a consequence of the action of the therapeutic agent,angiogenesis is prevented, reduced or inhibited in the subject. Morespecifically, the therapeutic agents can be used in the treatment of thedisease states and conditions of the angiogenic disease processes asdescribed below. More specifically, the LXA₄ and ATL therapeuticcompounds described throughout the specification can be used for thetreatment of restenosis, solid tumor tissue growth, neovascularization,e.g., retinal tissue, and reducing blood supply to tissue required tosupport new growth of tissue in a subject.

In another aspect, the present invention also pertains to methods forthe prevention, reduction or inhibition of angiogenesis in tissue of asubject. The method is accomplished by the administration of aneffective amount of an aspirin triggered lipoxin (ATL) (15-epi-LXA₄,such as 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ (ATL-1)), andpharmaceutically acceptable salts, esters, amides or prodrugs thereof,to a subject in need thereof. As a consequence of the action of thetherapeutic agent, angiogenesis is prevented or inhibited in thesubject.

The invention thus provides for a method for the general inhibition ofangiogenesis in tissue, and thereby inhibits or prevent events in thetissue which depend upon angiogenesis. Generally, the method comprisesadministering to the tissue a composition comprising anangiogenesis-inhibiting amount of, for example, ATL-1, LXA₄, 15-epi-LXA₄or 15-R/S-methyl, LXA₄.

As used herein, the term “angiogenesis” means the formation of new bloodvessels into a tissue or organ. Under normal physiological conditions,humans or animals only undergo angiogenesis in very specific restrictedsituations. For example, angiogenesis is associated with wound healing,fetal and embryonal development and formation of the corpus luteum,endometrium and placenta. The biochemical aspects of angiogenesis areassociated with a highly regulated system of angiogenic stimulators andinhibitors. Controlled angiogenesis has been found to be altered incertain disease states and, in many cases, the pathological damageassociated with the disease is related to the uncontrolled angiogenesis.

The bioprocesses of controlled and uncontrolled angiogenesis are thoughtto occur in a similar manner. Endothelial cells and pericytes,surrounded by a basement membrane, form capillary blood vessels. Erosionof the basement membrane promotes angiogenesis by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then break through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form an offshoot fromthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial offshoots can merge with each other to formcapillary loops, creating a new blood vessel. In the disease state,prevention of angiogenesis could avert the damage caused by the invasionof the new microvascular system.

Persistent, unregulated angiogenesis can occur in a multiplicity ofdisease states, tumor metastasis and abnormal growth by endothelialcells and supports the pathological damage seen in these conditions. Thediverse pathological states created due to unregulated angiogenesis havebeen grouped together as angiogenic dependent or angiogenic associateddiseases. The present invention provides therapies that are directed tocontrol the angiogenic processes thus leading to the abrogation ormitigation of these diseases. With the exception of traumatic woundhealing, corpus leuteum formation and embryogenesis, it is believed thatangiogenesis processes are associated with undesired, and often lifethreatening, disease processes and therefore, the use of the presenttherapeutic methods are selective for the disease, i.e., angiogenesis,and do not have deleterious side effects.

The following angiogenic diseases can be treated according to thepresent invention by use of the afore-mentioned ATLs, such as ATL-1 orLXA₄s such as 15-R/S-methyl, LXA₄. These angiogenic diseases include,but are not limited to, the following:

One example of a disease mediated by angiogenesis is ocular neovasculardisease. This disease is characterized by invasion of new blood vesselsinto the structures of the eye such as the retina or cornea. It isperhaps, one of the most common causes of blindness and is involved inover twenty eye diseases. For example, in age-related maculardegeneration, the associated visual problems are caused by an ingrowthof chorioidal capillaries through defects in Bruch's membrane withproliferation of fibrovascular tissue beneath the retinal pigmentepithelium. Angiogenic damage is also associated with diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma and retrolental fibroplasia. Other diseasesassociated with corneal neovascularization include, but are not limitedto, Vitamin A deficiency, contact lens overwear, atopic keratitis,superior limbic keratitis, epidemic keratoconjunctivitis, pterygiumkeratitis sicca, sjogrens, acne rosacea, lipid degeneration, chemicalburns, bacterial ulcers, fungal ulcers, phylectenulosis, syphilis,Mycobacteria infections, Herpes simplex infections, Herpes zosterinfections, Wegeners sarcoidosis, Scleritis, Steven's Johnson syndrome,periphigoid radial keratotomy, protozoan infections, Kaposi sarcoma,Mooren ulcer, Terrien's marginal degeneration, marginal keratolysis,rheumatoid arthritis, systemic lupus, polyarteritis, trauma, and cornealgraph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, vein occlusion, arteryocclusion, carotid obstructive disease, chronic uveitis/vitritis,mycobacterial infections, Lyme's disease, systemic lupus erythematosis,macular degeneration, sickle cell anemia, sarcoid, syphilis,pseudoxanthoma elasticum, Pagets disease, retinopathy of prematurity,Eales disease, Bests disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, Bechets disease, infections causing a retinitis orchoroiditis, presumed ocular histoplasmosis, trauma and post-lasercomplications. Other diseases include, but are not limited to, diseasesassociated with rubeosis and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreoretinopathy.

An even more prevalent disease in which angiogenesis is believed to beinvolved is rheumatoid arthritis. For example, the blood vessels in thesynovial lining of the joints undergo angiogenesis. In addition toforming new vascular networks, the endothelial cells release factors andreactive oxygen species that lead to pannus growth and cartilagedestruction. It is believed that the factors involved in angiogenesiscan actively contribute to, and help maintain, the chronically inflamedstate of rheumatoid arthritis.

It is believed that factors associated with angiogenesis can also have arole in osteoarthritis. The activation of the chondrocytes byangiogenic-related factors contributes to the destruction of the joint.At a later stage, the angiogenic factors can promote new bone formation.The present invention provides therapeutic intervention that preventsthe bone destruction and can halt the progress of the disease andprovide relief for persons suffering with arthritis.

Both ulcerative colitis and Crohn's disease are known to havehistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, can result in a chronic stage that ischaracterized by proliferation of vascular endothelial cells. An evenmore insidious pathological role associated with angiogenesis is foundin arteriosclerosis. The plaquing of the lumen of blood vessels has beenshown to have angiogenic stimulatory activity.

A frequent angiogenic disease of childhood is hemangioma. Generally, thetumors associated with the disease are benign and regress withoutintervention. In more severe cases, the tumors grow and create clinicalcomplications. Systemic forms of hemangiomas, the hemangiomatoses, havea high mortality rate. Therapy-resistant hemangiomas exist that cannotbe treated with therapeutics currently in use.

Angiogenesis is also responsible for damage found in hereditary diseasessuch as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. These diseases are characterized by multiple smallangiomas, tumors of blood or lymph vessels. The angiomas are found inthe skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding and sometimes with pulmonaryor hepatic arteriovenous fistula.

Of great concern is the disease state(s) associated with cancer(s).Often times, the cancer is associated with angiogenesis and isidentified by solid tumor formation and metastasis. Angiogenic factorsare associated with several solid tumors such as neuroblastoma,rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, and osteosarcoma. Itis known that a tumor cannot expand without a blood supply to providenutrients and remove cellular wastes. Tumors in which angiogenesis isimportant include solid tumors, and benign tumors such as acousticneuroma, neurofibroma, trachoma and granulomas. Prevention or inhibitionof angiogenesis could prevent or halt the growth of these tumors and thesubsequent degenerative condition due to the presence of the tumor.

Angiogenesis has also been associated with blood-born tumors includingleukemias, any of the various acute or chronic neoplastic diseases ofbone marrow in which unrestrained proliferation of white blood cellsoccurs, usually accompanied by anemia, impaired blood clotting, andenlargement of the lymph nodes, liver, and spleen. It is believed thatangiogenesis is significant as a caustive factor in the abnormalities inthe bone marrow that give rise to leukemia-like tumors.

Angiogenesis is important in two stages of tumor metastasis. The firststage where angiogenesis stimulation is important is in thevascularization of the tumor which allows tumor cells to enter the bloodstream and to circulate throughout the body. Once the tumor cells leavethe primary site, and find a secondary metastasis site, angiogenesismust occur before the new tumor can grow and expand. Therefore,prevention of angiogenesis could prevent metastasis of tumors andcontain the neoplastic growth at the primary site.

In a related embodiment, the present invention can be used incombination with other therapies such as conventional chemotherapydirected against solid tumors and metastases. ATLs, such as ATL-1 orLXA₄s, such as 15-R/S-methyl, LXA₄, can be administered during or afterchemotherapy. In a preferred embodiment, the drug should be administeredwhen the tumor tissue is responding to the toxic assault when vasculartissue is being reorganized to supply blood and nutrients to the tumortissue. Additionally, the use of ATLs, such as ATL-1 or LXA₄s can beused as a phrophylatic treatment after surgical removal of a tumor toprevent angiogenesis from occurring at the treatment site.

Knowledge of the role of angiogenesis in the maintenance and metastasisof tumors has led to a prognostic indicator for breast cancer. Theamount of neovascularization found in the primary tumor was determinedby counting the microvessel density in the area of the most intenseneovascularization in invasive breast carcinoma. A high level ofmicrovessel density was found to correlate with tumor recurrence.Control of angiogenesis by therapeutic means could possibly lead tocessation of the recurrence of the tumors.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimentalside effect of surgical procedures and can be caused or exacerbated byangiogenesis. Adhesions are a frequent complication of surgery and leadto problems such as small bowel obstruction.

Restenosis is a process of smooth muscle cell (SMC) migration andproliferation at the site of percutaneous transluminal coronaryangioplasty which hampers the success of angioplasty. The migration andproliferation of SMC's during restenosis can be considered a process ofangiogenesis which is inhibited by the present methods. Therefore, theinvention also contemplates inhibition, reduction or prevention ofrestenosis by inhibiting, reducing or preventing angiogenesis accordingto the present methods in a subject following angioplasty procedures.For inhibition or prevention of restenosis, an ATL, such as ATL-1 or anLXA₄, such as 15-R/S-methyl, LXA₄, can be administered, preferably viaintravenous injection, several days before the operation or after theangioplasty procedure for from about 2 to about 28 days, and moretypically for about the first 14 days following the procedure.

The term “subject” as used herein refers to any living organism in whichan angiogenic response is elicited. The term subject includes, but isnot limited to, humans, nonhuman primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, sheep, pigs, goatsand horses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

The term “mammal” as used herein refers to a living organism capable ofeliciting an immune response to an antigen. The term subject includes,but is not limited to, nonhuman primates such as chimpanzees and otherapes and monkey species, sheep, pigs, goats, horses, dogs, cats, mice,rats and guinea pigs, and the like.

The term “pharmaceutically acceptable salts, esters, amides, andprodrugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use ofthe compounds of the invention. The term “salts” refers to therelatively non-toxic, inorganic and organic acid addition salts ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds or byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.These can include cations based on the alkali and alkaline earth metals,such as sodium, lithium, potassium, calcium, magnesium and the like, aswell as non-toxic ammonium, quaternary ammonium, and amine cationsincluding, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. (See, for example, Berge S. M.,et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977;66:1-19 which isincorporated herein by reference).

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the parent compound of the above formulae, for example, byhydrolysis in blood. A thorough discussion is provided in T. Higuchi andV. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S.Symposium Series, and in Bioreversible Carriers in Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987, both of which are hereby incorporated by reference. As usedherein, a prodrug is a compound that, upon in vivo administration, ismetabolized or otherwise converted to the biologically, pharmaceuticallyor therapeutically active form of the compound. To produce a prodrug,the pharmaceutically active compound is modified such that the activecompound will be regenerated by metabolic processes. The prodrug can bedesigned to alter the metabolic stability or the transportcharacteristics of a drug, to mask side effects or toxicity, to improvethe flavor of a drug or to alter other characteristics or properties ofa drug. By virtue of knowledge of pharmacodynamic processes and drugmetabolism in vivo, once a pharmaceutically active compound isidentified, those of skill in the pharmaceutical art generally candesign prodrugs of the compound [see, e.g., Nogrady (1985) MedicinalChemistry A Biochemical Approach, Oxford University Press, New York,pages 388-392]. Conventional procedures for the selection andpreparation of suitable prodrug derivatives are described, for example,in “Design of Prodrugs,” ed. H. Bundgaard, Elsevier, 1985. Suitableexamples of prodrugs include methyl, ethyl and glycerol esters of thecorresponding acid.

The compounds of the invention can be formulated into pharmaceuticalcompositions as described, vide infra. In a preferred embodiment, thecompound can be administered over an extended period of time in asustained release composition. Sustained release compositions are knownin the art and one skilled in the art can formulate an acceptablecomposition based on generally recognized parameters in the art. In amost preferred embodiment, the glycerol ester can be used in thetreatment of inflammatory conditions, described herein, in sustainedrelease compositions, i.e., a transdermal patch, as known in the art.Suitable methods to prepare a transdermal patch can be found in U.S.Pat. Nos., 5,814,599, 5,846,974 or 4,201,211, the contents of which areincorporated herein by reference. More particularly, the compounds canbe delivered transdermally using the types of patch technologiesavailable from Ciba-Geigy Corporation and Alza Corporation. Theadministration of the pharmaceutical compositions of the presentinvention can be intermittent, or at a gradual, continuous, constant orcontrolled rate to a warm-blooded animal, such as a human being. Inaddition, the time of day and the number of times per day that thepharmaceutical formulation is administered can vary. Administrationpreferably is such that the active ingredients of the pharmaceuticalformulation interact with the inflammatory condition.

A “therapeutically effective amount” is an amount of an ATL, such asATL-1 or an LXA₄, such as 15-R/S-methyl, LXA₄, sufficient to produce ameasurable inhibition, reduction or prevents angiogenesis in the tissuebeing treated, i.e., an angiogenesis-inhibiting amount. Inhibition ofangiogenesis can be measured in situ by immunohistochemistry or by othermethods known to one skilled in the art such as measurement by FACanalysis that monitors P-selectin or VEGF receptors. See also FIGS. 4and 5.

More specifically, the pharmaceutical compositions of the invention caninclude a “therapeutically effective amount” or a “prophylacticallyeffective amount” of an antiangiogenic of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, e.g., a diminishment, reduction or prevention ofangiogenic factors associated with various disease states or conditions.A therapeutically effective amount of the antiangiogenic can varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the antiangiogenic to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the antibody orantibody portion are outweighed by the therapeutically beneficialeffects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result, i.e. prevent. Typically, since a prophylactic doseis used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antiangiogenic of the inventionis 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted thatdosage values can vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

The antiangiogenic compounds of the invention, e.g., an ATL, such asATL-1 or an LXA₄, such as 15-R/S-methyl, LXA₄, can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises an antiangiogenic ofthe invention and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In some instances, it can be beneficial to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers can further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of theantiangiogenic.

Additionally, the mono-, di- and/or tri-protected alcohols of thecompounds of the invention provide for sustained release of theinhibitory/preventative compound. For example, the tri-acyl analogs ofthe invention provide for such a sustained release of the hydrolyzedtri-hydroxy alcohol(s). The triacylated analogs are thus de-esterifiedin the blood of the subject.

The antiangiogenics of the invention can be incorporated into apharmaceutical composition suitable for parenteral administration. Othersuitable buffers include but are not limited to, sodium succinate,sodium citrate, sodium phosphate or potassium phosphate. Sodium chloridecan be used to modify the toxicity of the solution at a concentration of0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectantscan be included for a lyophilized dosage form, principally 0-10% sucrose(optimally 0.5-1.0%). Other suitable cryoprotectants include trehaloseand lactose. Bulking agents can be included for a lyophilized dosageform, principally 1-10% mannitol (optimally 2-4%). Stabilizers can beused in both liquid and lyophilized dosage forms, principally 1-50 mML-Methionine (optimally 5-10 mM). Other suitable bulking agents includeglycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally0.005-0.01%). Additional surfactants include but are not limited topolysorbate 20 and BRU surfactants.

The compositions of this invention can be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans. The preferred mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In a preferred embodiment, theantiangiogenic is administered by intravenous infusion or injection. Inanother preferred embodiment, the antiangiogenic is administered byintramuscular or subcutaneous injection. In the most preferredembodiment, the antiangiogenic is administered orally.

Alternatively, a preferred embodiment includes the use of the compoundsof the invention in eye-drop solutions. This provides for application tothe ease to inhibit or prevent ocular diseases such as glaucoma.Generally, the active ingredient, i.e., the compounds of the invention,would be dissolved in an aqueous solution that can be applied directlyto the eye.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antigen, antibody or antibody portion) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile, lyophilized powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andspray-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The antiangiogenics of the present invention can be administered by avariety of methods known in the art. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound can be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antiangiogenic of the invention can be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) canalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it can be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

The present invention also provides for packaged pharmaceuticalcompositions useful in the prevention or inhibition of angiogenicactivity in a subject. The packaged pharmaceutical composition includesa container holding a therapeutically effective amount of at least oneATL, such as ATL-1 or an LXA₄, such as 15-R/S-methyl, LXA₄, or apharmaceutically acceptable salt, esters, amide, or prodrug thereof andinstructions for using the therapeutic compound for preventing, reducingor inhibiting angiogenic activity in the subject. Additionally, thepresent invention provides therapeutically effective amounts of packagedpharmaceutical compositions, e.g., ATL-1, LXA₄, 15-epi-LXA₄ or15-R/S-methyl, LXA₄ or pharmaceutically acceptable salts, esters,amides, and prodrugs thereof, and instructions useful to treat, i.e.,inhibit or prevent, solid tumor tissue growth from undergoingneovascularization, neovascularization from occurring,neovascularization from occurring in retinal tissue, restenosis fromoccurring following angioplasty in a tissue wherein smooth muscle cellmigration occurs, or reducing blood supply to a tissue required tosupport new growth of new angiogenic tissue.

The present invention also provides angeogenic compounds that facilitateangiogenesis. Surprisingly, configurational isomers of LXA₄, LXA₄analogs and ATL analogs, LXB₄ and LXB₄ analogs and pharmaceuticallyacceptable salts, esters, amides or prodrugs thereof, provide theopposite effects with regard to revascularization of tissue by theabove-identified compounds of the invention. That is, it has beensurprisingly discovered that LXB₄ and LXB₄ analogs have the ability tostimulate regeneration and ingrowth of vascular or epithelial tissue intissues that are in need of such stimulation. This is especiallyimportant in tissue grafting, tissue engineering and prosthetic groupsites of attachment. Therefore, the present invention provides methodsof tissue regeneration, compounds for such application and packagedpharmaceuticals to accomplish such results.

For example, cardiovascular disease occurs as a consequence of thepartial or complete blockage of vessels carrying blood in the coronaryvascular system and in peripheral vasculature. Occlusion of the vesselcan results in death of tissue previously nourished by the occludedvessels or inability of the vessels to transport sufficient blood supplyto regions requiring high blood consumption and accompanying nutrients.Blood vessel occlusion can be partially compensated by the naturalprocess of angiogenesis, in which new conduits are formed to replace thefunction of the impaired vessels. These new conduits are referred to as“collateral” vessels and can help in the restoration of blood flow tothe deprived tissue, thereby constituting natural bypasses around theoccluded vessels. However, some individuals for various reasons areunable to generate sufficient collateral vessels to manage theconsequences of diminished blood flow from cardiovascular disease.

The LXB₄ compounds of the invention can be used to enhance the body'snatural ability to repair itself by undergoing natural angiogenesis. Ascan be seen from the contents of the specification and Figures, vesselgrowth is stimulated by these unique compounds. This process and use ofthe present compounds can be utilized for the treatment of wounds. TheLXB₄ compounds help stimulate the healing process, causingre-epithelialization and vascularization to occur.

Suitable lipoxin analogs, including ATLs, LXA₄s and LXB₄s encompassed bythe present invention include those having the followingcharacteristics.

The instant lipoxins comprising an “active region” and a “metabolictransformation region” as both terms are defined herein are generally ofthe following structure:wherein R₁ can be

wherein R₁ can be

and R₂ can be

In one embodiment, the lipoxin analogs of this invention have thefollowing structural formula I:

wherein X is R₁, OR.₁, or SR₁;

wherein R₁ is

(i) a hydrogen atom;

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straightchain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) an aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which can be a straight chain or        branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms,inclusive;

wherein Q₁ is (C═O), SO₂ or (CN);

wherein Q₃ is O, S or NH;

wherein one of R₂ and R₃ is a hydrogen atom and the other is

(a) a hydrogen atom;

(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straightchain or branched;

(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straightchain or branched; or

(e) R_(a) Q₂ R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched; and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which can be straight chain or branched;

wherein R₄ is

(a) a hydrogen atom; (b) an alkyl of 1 to 6 carbon atoms, inclusive,which can be straight chain or branched;

wherein Y₁ or Y₂ is —OH, methyl, or —SH and wherein the other is

(a) a hydrogen atom

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3; and

each Z, independently, is a cyano, a nitro, or a halogen atom;

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched; or

(d) an alkoxy of 1 to 4 carbon atoms, inclusive;

or Y₁ and Y₂ taken together are

(a) ═NH; or

(b) ═O;

wherein R₅ is

(a) an alkyl of 1 to 9 carbon atoms which can be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) a phenyl; or

(iii) substituted phenyl

-   -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which can be a straight chain or        branched, and hydroxyl;

(c) R_(a)Q_(a)R_(b)

wherein Q_(a) is O or S;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which can bestraight chain or branched;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are each, independently:

(i) a hydrogen atom;

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3, and wherein each Z,independently, is a cyano, a nitro, or a halogen atom;

(e) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched; and

wherein R₆ is

(a) a hydrogen atom;

(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain orbranched;

(c) a halogen.

In one embodiment of this invention, the lipoxin analogs have thefollowing structure II:

wherein X is R₁, OR₁, or SR₁; wherein R₁ is

(i) a hydrogen atom;

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straightchain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) an aralkyl of 7 to 12 carbon atoms;

(v) a phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule, such as but not limited tofluorescent labels; or

(viii) an alkenyl of 2 to 8 carbon atoms, inclusive, straight chain orbranched;

wherein Q₁ is (C═O), SO₂ or (C═N);

wherein Q₃ is O, S or NH;

wherein one of R₂ and R₃ is hydrogen and the other is

(a) a hydrogen atom;

(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straightchain or branched;

(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straightchain or branched; or (e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which can bestraight chain or branched;

wherein R₄ is

(a) a hydrogen atom;

(b) alkyl of 1 to 6 carbon atoms, inclusive, which can be straight chainor branched;

wherein Y₁ or Y₂ is —OH, methyl, —H or —SH and wherein the other is

(a) a hydrogen atom;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3

wherein each Z, independently, is a cyano, a nitro, or a halogen atom;

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched;

(d) an alkoxy of 1 to 4 carbon atoms, inclusive; or Y₁ and Y₂ takentogether are

(a) ═NH; or

(b) ═O;

wherein R₅ is

(a) an alkyl of 1 to 9 carbon atoms which can be straight chain orbranched;

(b) —(CH₂), —R_(i)

wherein n=0 to 4 and R_(i) is

(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(c) —R_(a)Q_(a)R_(b)

wherein Q_(a) is —O— or —S—; and

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which can bestraight chain or branched;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are each independently:

(i) a hydrogen atom; or

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3

wherein each Z, independently, is a cyano, a nitro, or a halogen atom,

(e) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched.

In one embodiment of this invention, the lipoxin analogs have thefollowing structure III:

wherein X is R₁, OR₁, or SR₁; wherein R₁ is

(i) a hydrogen atom;

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straightchain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) an aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) an alkenyl of 2 to 8 carbon atoms, inclusive, straight chain orbranched;

wherein Q₁ is (C═O), SO₂ or (C═N);

wherein Q₃ is O, S or NH;

wherein one of R₂ and R₃ is hydrogen atom and the other is

(a) a hydrogen atom;

(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straightchain or branched;

(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which can bestraight chain or branched;

wherein R₄ is

(a) a hydrogen atom; or

(b) an alkyl of 1 to 6 carbon atoms, inclusive, which can be straightchain or branched;

wherein Y₁ or Y₂ is hydroxyl, methyl, hydrogen or thiol and

wherein the other is

(a) a hydrogen atom;

(b) CH_(a)Z_(b)

where a+b=3, a 0 to 3, b=0 to 3

wherein each Z, independently, is a cyano, a nitro, or a halogen atom;

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched;

(d) an alkoxy of 1 to 4 carbon atoms, inclusive; or Y₁ and Y₂ takentogether are

(a) ═NH; or

(b) ═O; and

wherein R₅ is

(a) an alkyl of 1 to 9 carbon atoms which can be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl;

(iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(′O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(c) R_(a)Q_(a)R_(b)

wherein Q_(a) is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which can bestraight chain or branched; or

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are each independently:

(i) a hydrogen atom; or

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3

wherein each Z, independently, is a cyano, a nitro, or a halogen atom,

(e) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched.

In another embodiment of this invention, lipoxin analogs have thefollowing structural formula IV:

wherein X is R₁, OR₁, or SR₁; wherein R₁ is

(i) a hydrogen atom;

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straightchain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) an aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) an alkenyl of 2 to 8 carbon atoms, inclusive, straight chain orbranched;

wherein Q₁ is (C═O), SO₂ or (CN);

wherein Q₃ is O, S or NH;

wherein one of R₂ and R₃ is hydrogen and the other is

(a) a hydrogen atom;

(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straightchain or branched;

(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which can bestraight chain or branched;

wherein R₄ is

(a) a hydrogen atom; or

(b) an alkyl of 1 to 6 carbon atoms, inclusive, which can be straightchain or branched;

wherein Y₁ or Y₂ is —OH, methyl, or —SH and wherein the other is

(a) a hydrogen atom;

(b) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0 to 3, wherein each Z,independently, is a cyano, a nitro, or a halogen atom;

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched; or

(d) an alkoxy of 1 to 4 carbon atoms, inclusive;

or Y₁ and Y₂ taken together are

(a) ═NH; or

(b) ═O;

wherein R₅ is

(a) an alkyl of 1 to 9 carbon atoms which can be straight chain orbranched;

(b) —(CH₂)_(n) 13 R_(i)

wherein n=0 to 4 and R_(i) is

(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(c) R_(a)Q_(a)R_(b)

wherein Q_(a) is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which can bestraight chain or branched;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are each independently:

(i) a hydrogen atom; or (ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3and

wherein each Z, independently, is a cyano, a nitro, or a halogen atom;or

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched; and

wherein R₆ is

(a) a hydrogen atom;

(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain orbranched; or

(c) a halogen atom.

In another embodiment of this invention, lipoxin analogs have thefollowing structural formula V:

wherein R₁ is

(i) a hydrogen atom;

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straightchain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) an aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) an alkenyl of 2 to 8 carbon atoms, inclusive, straight chain orbranched;

wherein n=1 to 10, inclusive;

wherein R₂, R_(3a), and R_(3b) are each independently:

(a) a hydrogen atom;

(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straightchain or branched;

(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched; and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which can be straight chain or branched;

wherein Y₁ or Y₂ is —OH, methyl, hydrogen, or —SH and

wherein the other is

(a) a hydrogen atom;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3, and

wherein each Z, independently, is a cyano, a nitro, or a halogen atom;

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched;

(d) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain orbranched;

or Y₁ and Y₂ taken together are

(a) ═NH; or

(b) ═O;

wherein Y₃ or Y₄ is —OH, methyl, hydrogen, or —SH and

wherein the other is

(a) a hydrogen atom;

(b) CH_(a)Z_(b)

wherein a+b=3, a=0 to 3, b=0 to 3,

and wherein each Z, independently, is a cyano, a nitro, or a halogenatom;

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched;

(d) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain orbranched;

or Y₃ and Y₄ taken together are

(a) ═NH; or

(b) ═O;

wherein Y₅ or Y₆ is —OH, methyl, hydrogen, or —SH and

wherein the other is

(a) a hydrogen atom;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3

wherein each Z, independently, is a cyano, a nitro, or a halogen atom;

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched;

(d) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain orbranched;

or Y₅ and Y₆ taken together are

(a) ═NH; or

(b) ═O;

wherein R₅ is

(a) an alkyl of 1 to 9 carbon atoms which can be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(c) —R_(a)Q_(a)R_(b)

wherein Q_(a) is —O— or —S—; and

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is either alkyl of 0 to 8 carbon atoms, inclusive, whichcan be straight chain or branched or substituted phenyl;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are each independently:

(i) a hydrogen atom; or

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3, and

wherein each Z, independently, is a cyano, a nitro, or a halogen atom;or

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched.

In another embodiment of this invention, lipoxin analogs have thestructural formula VI:

wherein R_(a) is

(a) a hydrogen atom; or

(b) alkyl of 1 to 8 carbon atoms;

wherein R_(x) is

(a) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(b) a substituted phenoxy

wherein Z_(i) through Z_(v) are as defined above; or

wherein Z_(i) through Z_(v) are as defined above.

In another preferred embodiment of this invention, lipoxin analogs havethe following structural formula VII:

wherein R_(a) is

(a) a hydrogen atom; or

(b) an alkyl of 1 to 8 carbon atoms;

wherein R_(b) and R_(c) are each independently:

(a) a hydrogen atom;

(b) a hydroxyl, or a thiol;

(c) a methyl or a halomethyl;

(d) a halogen;

(e) an alkoxy of 1 to 3 carbon atoms;

wherein R_(d) and R_(e) are each independently:

(a) a hydrogen atom;

(b) a hydroxyl, or thiol;

(c) a methyl or halomethyl;

(d) a halogen;

(e) an alkoxy of 1 to 3 carbon atoms; or

(f) an alkyls or haloalkyl of 2 to 4 carbon atoms, inclusive, which canbe straight chain or branched.

In another preferred embodiment of this invention, the lipoxin analogshave the structural formula VIII:

wherein R_(a) is

(a) a hydrogen atom; or

(b) an alkyl of 1 to 8 carbon atoms;

wherein R_(b) and R_(c) are each independently:

(a) a hydrogen atom;

(b) a hydroxyl or a thiol;

(c) a halomethyl;

(d) a halogen;

(e) an alkyl of 1 to 3 carbon atoms, inclusive, straight chain orbranched; or

(f) an alkoxy of 1 to 3 carbon atoms, inclusive;

wherein R_(d) and R_(e) are each independently:

(a) a hydrogen atom;

(b) a hydroxyl, or a thiol;

(c) a methyl or a halomethyl;

(d) a halogen;

(e) an alkoxy of 1 to 3 carbon atoms, inclusive; or

(f) an alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive, which canbe straight chain or branched.

In another embodiment of this invention, the lipoxin analogs have thestructural formula IX:

wherein R_(a) is

(a) a hydrogen atom; or

(b) an alkyl of 1 to 8 carbon atoms;

wherein R_(b) and R_(c) are each independently:

(a) a hydrogen atom;

(b) a hydroxyl or thiol;

(c) a halomethyl;

(d) a halogen;

(e) an alkyl of 1 to 3 carbon atoms, inclusive, straight chain orbranched;

(f) an alkoxy of 1 to 3 carbon atoms, inclusive; and

wherein R₅ is

(a) an alkyl of 1 to 9 carbon atoms which can be straight chain orbranched;

(b) —(CH₂)_(n), —R_(i)

wherein n=0 to 4 and R_(i) is

(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(c) R_(a)Q_(a)R_(b)

wherein Q_(a) is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is either alkyl of 0 to 8 carbon atoms, inclusive, whichcan be straight chain or branched or substituted phenyl;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are each, independently:

(i) a hydrogen atom; or

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3

wherein each Z, independently, is a cyano, a nitro, or a halogen atom;or

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched.

In another preferred embodiment, the compounds have the structuralformula X:

wherein R_(a) is

(a) a hydrogen atom; or

(b) alkyl of 1 to 8 carbon atoms, inclusive, straight chain or branched;and

wherein R_(b) and R_(c) are each, independently:

(a) a hydrogen atom;

(b) a hydroxyl or a thiol;

(c) a halomethyl;

(d) a halogen;

(e) an alkyl of 1 to 3 carbon atoms, inclusive, straight chain orbranched;

(f) an alkoxy of 1 to 3 carbon atoms, inclusive.

In another preferred embodiment, the compounds have the structuralformula XI:

wherein R_(a) is

(i) a hydrogen atom;

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straightchain or branched; or

(iii) a detectable label molecule;

wherein n=1 to 10, inclusive;

wherein Y₂, R_(3a), and R_(3b) are each, independently:

(a) a hydrogen atom;

(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straightchain or branched;

(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched; and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which can be straight chain or branched;

wherein Y₁ is —OH, methyl, or —SH;

wherein Y₂ is

(a) a hydrogen atom;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3

wherein each Z, independently, is a cyano, a nitro, or a halogen atom;or

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched;

wherein Y₃ and Y₅ are each independently:

(a) a hydrogen atom;

(b) CH_(a)Z_(b)

wherein a+b=3, a=0 to 3, b=0 to 3 and wherein each Z, independently, isa cyano, a nitro, or a halogen atom; or

(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched;

wherein Y₄ and Y₆ are each, independently

(a) a hydrogen atom;

(b) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain orbranched;

(c) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain orbranched; or

(d) a hydroxyl or thiol; and

wherein R₅ is

(a) an alkyl of 1 to 9 carbon atoms which can be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 3 and R_(i) is

(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl;

(iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(c) —R_(a)Q_(a)R_(b)

wherein Q_(a) is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which canbe straight chain or branched;

wherein R_(b) is

(a) a substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl;

(b) a substituted phenoxy

wherein Z_(i) through Z_(v) are as defined above; or

wherein Z_(i) through Z_(v) are as defined above;

(d) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched.

In certain embodiments of this invention, the compounds of thisinvention have the following structural formulas:

where R′ is H or CH₃; and where the substituents at C* are in the Rconfiguration.

In other preferred embodiments of this invention, the compounds of thisinvention have the following structural formulas:

where the substituents at the C* are in the R configuration.

It is to be understood that the carboxylic acids and esters of theinvention can be converted, if necessary, into pharmaceuticallyacceptable salts.

In certain embodiments of the invention, LXB₄ or the C5 and C14 and C15alkanoates (acetates) of LXB₄ may be excluded.

Lipoxins Having Phenoxy or Thiophenoxy Substituents

In another aspect, lipoxins and lipoxin analogs useful as therapeuticagents in the treatment of the maladies, disease states or conditionsdescribed throughout the specification has the formula:

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbon atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein Q₃ and Q₄ are each independently O, S or NH;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbon atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,        inclusive, straight chain or branched, an alkoxy of 1 to 4        carbon atoms, inclusive, or CH_(a)Z_(b) where a+b=3, a=0 to 3,        b=0 to 3 and Z is cyano, nitro or a halogen;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereof.

In yet another aspect, lipoxins and lipoxin analogs useful astherapeutic agents in the treatment of the maladies, disease states orconditions described throughout the specification has the formula:

-   -   wherein X is R₁, OR_(x), or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbon atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbon atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,        inclusive, straight chain or branched, an alkoxy of 1 to 4        carbon atoms, inclusive, or CH_(a)Z_(b) where a+b=3, a=0 to 3,        b=0 to 3 and Z is cyano, nitro or a halogen;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereof.

In still another aspect, lipoxins and lipoxin analogs useful astherapeutic agents in the treatment of the maladies, disease states orconditions described throughout the specification has the formula:

-   -   wherein X is R₁, OR_(x), or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbon atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein one of R₂ and R₃ is a hydrogen atom and the other is        -   (a) H;        -   (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be            a straight chain or branched;        -   (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;        -   (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may            be straight chain or branched; or        -   (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is            alkylene of 0 to 6 carbon atoms, inclusive, which may be            straight chain or branched and wherein R_(b) is alkyl of 0            to 8 carbon atoms, inclusive, which may be straight chain or            branched, provided when R_(b) is 0, then R_(b) is a hydrogen            atom;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereof.

In yet another aspect, lipoxins and lipoxin analogs useful astherapeutic agents in the treatment of the maladies, disease states orconditions described throughout the specification has the formula:

-   -   wherein X is R₁, OR₁, or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbon atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then X        is absent;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is        wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group;    -   wherein R₆ is        -   (a) H;        -   (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight            chain or branched;

wherein T is O or S, and pharmaceutically acceptable salts thereof.

In one aspect, lipoxins and lipoxin analogs useful as therapeutic agentsin the treatment of the maladies, disease states or conditions describedthroughout the specification has the formula:

-   -   wherein X is R₁, OR_(x), or SR₁;    -   wherein R₁ is        -   (i) a hydrogen atom;        -   (ii) an alkyl of 1 to 8 carbon atoms, inclusive, which may            be straight chain or branched;        -   (iii) a cycloalkyl of 3 to 10 carbon atoms;        -   (iv) an aralkyl of 7 to 12 carbon atoms;        -   (v) phenyl;        -   (vi) substituted phenyl    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl;        -   (vii) a detectable label molecule; or        -   (viii) a straight or branched chain alkenyl of 2 to 8 carbon            atoms, inclusive;    -   wherein R₄ is        -   (a) H;        -   (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be            a straight chain or branched;    -   wherein R₅ is    -   wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each        independently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a        hydrogen atom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8        carbon atoms, inclusive, which may be a straight chain or        branched, and hydroxyl or a substituted or unsubstituted,        branched or unbranched alkyl group; and

pharmaceutically acceptable salts thereof.

In preferred embodiments, X is OR₁ wherein R₁ is a hydrogen atom, analkyl group of 1 to 4 carbon atoms or a pharmaceutically acceptablesalt, Q₁ is C═O, R₂ and R₃, if present, are hydrogen atoms, R₄ is ahydrogen atom or methyl, Q₃ and Q₄, if present, are both O, R_(6,) ifpresent, is a hydrogen atom, Y₁, if present, is OH, T is O and R₅ is asubstituted phenyl, e.g.,

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl. In certainembodiments for R₅, para-fluorophenyl and/or unsubstituted phenyl arepreferred, e.g., 15-epi-16-(para-fluoro)-phenoxy-LXA₄,16-(para-fluoro)-phenoxy-LXA₄, 15-epi-16-phenoxy-LXA₄ or16-phenoxy-LXA₄.

In still another aspect, the present invention is directed topharmaceutical compositions including compounds having the formulaedescribed throughout the specification and a pharmaceutically acceptablecarrier. In one embodiment, a preferred compound is

In one embodiment, Q₁ is a carbonyl, X is a hydroxyl or an —OR, whereinR is an alkyl group, i.e., methyl or ethyl groups, and R₄ is a hydrogenatom.

In other embodiments, Y₁ is a hydroxyl and the carbon bearing thehydroxyl can have an R or S configuration. In most preferredembodiments, the chiral carbon bearing the hydroxyl group, e.g., Y₁, isdesignated as a 15-epi-lipoxin as is known in the art.

In certain embodiments the chirality of the carbons bearing the R₂, R₃,Q₃ and Q₄ groups can each independently be either R or S. In preferredembodiments, Q₃ and Q₄ have the chiralities shown in above-referencedstructures.

In preferred embodiments, R₄ is a hydrogen. In other preferredembodiments, R₆ is a hydrogen.

Additionally, R₅ can be a substituted or unsubstituted, branched orunbranched alkyl group having between 1 and about 6 carbon atoms,preferably between 1 and 4 carbon atoms, most preferably between 1 and3, and preferably one or two carbon atoms. The carbon atoms can havesubstituents which include halogen atoms, hydroxyl groups, or ethergroups.

The compounds useful in the present invention can be prepared by thefollowing synthetic scheme:

wherein X, Q₁, Q₃, Q₄, R₂, R₃, R₄, R₅, R₆, Y₁ and T are as definedabove. Suitable methods known in the art to can be used to produce eachfragment. For example, the acetylenic fragment can be prepared by themethods discussed in Nicolaou, K. C. et al. (1991) Angew. Chem. Int. Ed.Engl. 30:1100; Nicolaou, K. C. et al. (1989) J. Org. Chem. 54:5527;Webber, S. E. et al. (1988) Adv. Exp. Med. Biol. 229:61; and U.S. Pat.No. 5,441,951. The second fragment can be prepared by the methods ofRaduchel, B. and Vorbruggen, H. (1985) Adv. Prostaglandin ThromboxaneLeukotriene Res. 14:263. As a consequence, the acetylenic intermediatesare also encompassed by the present invention as being useful for thetreatments of the various maladies described herein. Similar approachescan be taken to produce LXB₄ acetylenic compounds as described in, forexample, U.S. Pat. No. 6,316,648.

A “lipoxin analog” shall mean a compound which has an “active region”that functions like the active region of a “natural lipoxin”, but whichhas a “metabolic transformation region” that differs from naturallipoxin. Lipoxin analogs include compounds which are structurallysimilar to a natural lipoxin, compounds which share the same receptorrecognition site, compounds which share the same or similar lipoxinmetabolic transformation region as lipoxin, and compounds which areart-recognized as being analogs of lipoxin. Lipoxin analogs includelipoxin analog metabolites. The compounds disclosed herein may containone or more centers of asymmetry. Where asymmetric carbon atoms arepresent, more than one stereoisomer is possible, and all possibleisomeric forms are intended to be included within the structuralrepresentations shown. Optically active (R) and (S) isomers may beresolved using conventional techniques known to the ordinarily skilledartisan. The present invention is intended to include the possiblediastereiomers as well as the racemic and optically resolved isomers.

The terms “corresponding lipoxin” and “natural lipoxin” refer to anaturally-occurring lipoxin or lipoxin metabolite. Where an analog hasactivity for a lipoxin-specific receptor, the corresponding or naturallipoxin is the normal ligand for that receptor. For example, where ananalog is a LXA₄ specific receptor on differentiated HL-60 cells, thecorresponding lipoxin is LXA₄. Where an analog has activity as anantagonist to another compound (such as leukotriene C4 and/orleukotriene D4), which is antagonized by a naturally-occurring lipoxin,that natural lipoxin is the corresponding lipoxin.

“Active region” shall mean the region of a natural lipoxin or lipoxinanalog, which is associated with in vivo cellular interactions. Theactive region may bind the “recognition site” of a cellular lipoxinreceptor or a macromolecule or complex of macromolecules, including anenzyme and its cofactor. For example, lipoxin A₄ analogs have an activeregion comprising C₅-C₁₅ of natural lipoxin A₄. Similarly, for example,lipoxin B₄ analogs have an active region comprising C5-C14 of naturallipoxin B₄.

The term “recognition site” or receptor is art-recognized and isintended to refer generally to a functional macromolecule or complex ofmacromolecules with which certain groups of cellular messengers, such ashormones, leukotrienes, lipoxins, must first interact before thebiochemical and physiological responses to those messengers areinitiated. As used in this application, a receptor may be isolated, onan intact or permeabilized cell, or in tissue, including an organ. Areceptor may be from or in a living subject, or it may be cloned. Areceptor may normally exist or it may be induced by a disease state, byan injury, or by artificial means. A compound of this invention may bindreversibly, irreversibly, competitively, noncompetitively, oruncompetitively with respect to the natural substrate of a recognitionsite.

The term “metabolic transformation region” is intended to refergenerally to that portion of a lipoxin, a lipoxin metabolite, or lipoxinanalog including a lipoxin analog metabolite, upon which an enzyme or anenzyme and its cofactor attempts to perform one or more metabolictransformations which that enzyme or enzyme and cofactor normallytransform on lipoxins. The metabolic transformation region may or maynot be susceptible to the transformation. Similarly, such regions arepossibly located within ATL and LXB₄ analogs. A nonlimiting example of ametabolic transformation region of a lipoxin is a portion of LXA₄ thatincludes the C-13,14 double bond or the C-15 hydroxyl group, or both.

The term “detectable label molecule” is meant to include fluorescent,phosphorescent, and radiolabeled molecules used to trace, track, oridentify the compound or receptor recognition site to which thedetectable label molecule is bound. The label molecule may be detectedby any of the several methods known in the art.

The term “labeled analog” is further understood to encompass compoundswhich are labeled with radioactive isotopes, such as but not limited totritium (³H), deuterium (²H), carbon (¹⁴C), or otherwise labeled (e.g.fluorescently). The compounds of this invention may be labeled orderivatized, for example, for kinetic binding experiments, for furtherelucidating metabolic pathways and enzymatic mechanisms, or forcharacterization by methods known in the art of analytical chemistry.

The term “inhibits metabolism” means the blocking or reduction ofactivity of an enzyme which metabolizes a native eicosanoid. Theblockage or reduction may occur by covalent bonding, by irreversiblebinding, by reversible binding which has a practical effect ofirreversible binding, or by any other means which prevents the enzymefrom operating in its usual manner on another lipoxin analog, includinga lipoxin analog metabolite, a lipoxin, or a lipoxin metabolite.Similarly, this also applies to ATL and LXB₄ analogs.

The term “resists metabolism” is meant to include failing to undergo oneor more of the metabolic degradative transformations by at least one ofthe enzymes which metabolize lipoxins, ATL or LXB₄ analogs. Twononlimiting examples of LXA₄ analog that resists metabolism are 1) astructure which can not be oxidized to the 15-oxo form, and 2) astructure which may be oxidized to the 15-oxo form, but is notsusceptible to enzymatic reduction to the 13,14-dihydro form.

The term “more slowly undergoes metabolism” means having slower reactionkinetics, or requiring more time for the completion of the series ofmetabolic transformations by one or more of the enzymes which metabolizelipoxin, lipoxin analogs, ATL or LXB₄ analogs. A nonlimiting example ofa LXA₄ analog which more slowly undergoes metabolism is a structurewhich has a higher transition state energy for C-15 dehydrogenation thandoes LXA₄ because the analog is sterically hindered at the C-16.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

The term “halogen” is meant to include fluorine, chlorine, bromine andiodine, or fluoro, chloro, bromo, and iodo.

The invention is further illustrated by the following examples which inno way should be construed as being further limiting. The contents ofall references, pending patent applications and published patentapplications, cited throughout this application, including thosereferenced in the background section, are hereby incorporated byreference. It should be understood that the models used throughout theexamples are accepted models and that the demonstration of efficacy inthese models is predictive of efficacy in humans.

Materials and Methods

Cell Culture

Human umbilical vein endothelial cells (HUVEC) were isolated by 0.1%collagenase digestion (Worthington Biochemical, Bedford, Mass.) andpropagated on gelatin-coated (0.1%) tissue culture plates in medium 199(Gibco BRL, Grand Island, N.Y.) supplemented with 20% heat-inactivatedfetal bovine serum (BioWhittaker, Walkersville, Md.), 50 μg/ml ofendothelial cell mitogen (Biomedical Technologies, Stoughton, Mass.), 8U/ml heparin (APP, Los Angeles, Calif.), 50 U/ml penicillin and 15 μg/mlstreptomycin. Only passages 2 and 3 were used in reported experiments.

Endothelial Cell Proliferation

HUVEC (5×10³) were plated in 96-well plates coated with 0.1% gelatin for1 h at room temperature. After 24 h, the medium was removed and replacedwith fresh medium 199 supplemented with 5% fetal bovine serum anddifferent concentrations of recombinant human VEGF₁₆₅ (R&D Systems,Minneapolis, Minn.), LTD₄ or LTB₄. Endothelial cells were enumeratedafter 72 h using the MTT® assay (Sigma, St. Louis, Mo.) (15). ATL-1 wasprepared as in Clish et al. (13). Percent inhibition was evaluated in asimilar manner and included a 15 minute incubation (37° C.) with15-epi-lipoxin A₄, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ methylester (ATL-1), LXA₄, 15-epi-LXA₄ or 15-R/S-methyl, LXA₄ prior to theaddition of agonists. All incubations were performed in triplicate.Before each experiment the integrity and concentration of ATL-1, LXA₄,15-epi-LXA₄ or 15-R/S-methyl, LXA₄ was assessed by physical methodsincluding LC/MS/MS and UV (13).

Endothelial Cell Migration

VEGF, ATL-1 or vehicle were added to the lower wells of a 48-wellchemotaxis chamber (NeuroProbe, Cabin John, Md.). The wells wereoverlaid with a 10 μm pore size polycarbonate filter coated with 0.1%gelatin. HUVEC (1×10⁶) were placed in the upper wells and the chamberwas incubated (37° C., 5% CO₂ for 12 h). Following incubations, filterswere removed, scraped of cells from the upper surface, fixed and stainedwith Diff-Quik (Dade Behring, Newark, Del.). Cells that migrated acrossthe filter toward the lower surface were enumerated by light microscopy;four fields were counted at high magnification (100×). Incubations wereperformed in triplicate. To assess inhibition, endothelial cells weresuspended in media with vehicle or ATL-1 for 15 min before placement inthe chamber.

Quantitative Determination of DNA Fragmentation

DNA fragmentation in individual apoptotic cells was quantitated using aphotometric enzyme immunoassay (Apotosis detection kit: R&D Systems).HUVECs grown in 96-well microtiter plates (5×10³ cells/well) wereincubated for 3 days, fixed with 3.7% formaldehyde, and permeabilizedwith proteinase K before the labeling. Biotinylated nucleotides areincorporated onto the DNA fragments and detected by usingsteptavidin-horseradish peroxidase conjugate followed by the substrateTACS-Sapphire.

Inflammatory Angiogenesis

Angiogenesis was assessed with murine air pouches that were raised viasubcutaneous injection of sterile air (3 ml) beneath the dorsal skin ofanesthetized mice (BALB/c, male 6-8 wk). After 24 hrs, ATL-1 (10μg/pouch) or vehicle was delivered locally, immediately before theinjection of VEGF (1 μg/pouch). The vascular content was assessed by theformation of vascular casts (as in 16). Briefly, mice were anesthetized(at 144 h) and peripheral vasodilation was raised by placing the animalsin a heated jacket (40° C., 10 min). Vascular casts were formed by thei.v. injection of 1 ml 5% carmine red (Sigma) in 5% gelatin solutionwarmed to 40° C. Air pouch linings were dissected and weighed. Thetissue was then dissolved in 2 ml of 3 N NaOH solution for 0.5 h, 21° C.and completely digested in hot water (56° C.) for 10 min. Digestedsamples were centrifuged (2500 rpm, 15 min) and filtered through a 0.45μm filter. The dye content was quantified employing a 96-well platespectrophotometer at 530 nm using a calibration curve. The results wereexpressed as vascular index (VI) as micrograms carmine dye/milligramweight of tissue, for n=4 animals/group. For visualization of thevasculature, the dorsal surface of the pouches was excised and fixed informalin for 48 h. The tissues were dehydrated with 100% ethanol (5days, 4° C.) and cleared by immersion in cedar wood oil for 2 weeks. Inanother set of experiments, mice were anesthetized and injected i.v.with 200 μl of 0.05 g/ml fluorescein isothiocyanate-dextran (Sigma) inPBS at 144 h immediately before sacrifice. Dissected linings were fixed,mounted on glass slides and examined for fluorescence (Nikon Eclipsemodel E600). In both protocols, the observers were not blinded to thetreatments.

Immunohistochemistry

Air pouch membranous tissues were fixed in 10% buffered formalinovernight and processed for paraffin embedding. Five-micrometer paraffinsections of membrane tissue cut on face were used forimmunohistochemistry for CD31 expression. Briefly, slides weredeparaffinized and pretreated in 0.25% trypsin (Sigma Chemical) for 20min at 37° C., followed by washing in distilled water. All further stepswere performed at room temperature in a hydrated chamber. Slides werepretreated with Peroxidase Block (DAKO, Carpinteria, Calif.) for 5 minto quench endogenous peroxidase activity, followed by a 1:5 dilution ofgoat serum in 50 Mm Tris-Cl, pH 7.4, for 20 min to block nonspecificbinding sites. Primary rat anti-murine CD31 antibody (BD PharMingen, SanDiego, Calif.) was applied at a 1:100 dilution in 50 mM Tris-Cl, pH 7.4,with 3% goat serum for 1 h. After washing in 50 mM Tris-Cl, pH 7.4,secondary rabbit anti-rat antibody (DAKO) was applied at a 1:200dilution in 50 mM Tris-Cl, pH 7.4, with 3% goat serum for 30 min. Slideswere washed again in 50 mM Tris-Cl, pH 7.4, and goat anti-rabbithorse-radish peroxidase-conjugated antibody (Envision detection kit:DAKO) was applied for 30 min. After further washing, immunoperoxidasestaining was developed using a DAB chromogen kit (DAKO) per themanufacturer and counterstained with hematoxylin.

Statistical Analysis

Results are presented as means±S.E.M. Statistical evaluation of theresults was performed by analysis of variance, and values of P<0.05 weretaken to represent statistically significant differences between groupmeans.

Results and Discussion

The aspirin-triggered lipoxin A₄ stable analog (denoted ATL-1) proved apotent inhibitor of VEGF-stimulated proliferation of HUVECs (IC₅₀ of ˜3nM) (FIG. 1). Inhibition was concentration-dependent and maximal plateauat 10 nM and was partially reversed (from 38.0±2.5 to 78.0±6.21% ofVEGF-stimulated proliferation, P<0.05) by incubating the cells withgenistein, an inhibitor of tyrosine kinase activity (50 μM, 5 min, 37°C., n=3). Even at high concentrations (100 nM), the ATL-1 analog alonehad no apparent actions on HUVEC proliferation (FIG. 1 inset). In sharpcontrast, LXB₄ stable analogs increased proliferation. Because these arerelated structures, the separate actions of ATL and LXB₄ analogs withthese cells indicate that the ATL-1 response is highly stereoselective.Also, a direct comparison of ATL-1 with native LXA₄ and 15-epi-LXA₄ atequimolar concentrations (10 nM) in a representative experiment showedthat ATL-1>15-epi-LXA₄>LXA₄ in rank order of activity (e.g., 63.3±3.3,59.8±1.8 and 38.1±2.0% inhibition, respectively). After exposure of thecells to ATL-1, ˜98% of the HUVEC remained viable, as determined bytrypan blue exclusion assay, indicating that the compound was notcytotoxic. In addition to being an endothelial cell-specific mitogen,VEGF is also an endothelial cell survival factor, thus promotingangiogenesis not only by stimulating cell proliferation but also byinhibiting endothelial cell apoptosis (17).

To determine whether this new inhibitory action of ATL-1 on HUVECproliferation involved apoptosis, DNA fragments were quantitated (underMaterials and Methods). Neither the ATL-1 (100 nM) alone nor incombination with VEGF (3 ng/ml) affected DNA fragmentation pattern (n=2, d=3), suggesting that the antiproliferative actions of the ATL-1analog were not a results of induction of apoptosis in endothelialcells. Binding of αv integrins by endothelial cells is accompanied by adecrease in the tumor-suppressor p53 activity and inhibition ofapoptotic pathways, thereby facilitating the formation of new bloodvessels (18). Along these lines, it was recently shown that p53 canup-regulate human 15-lipoxygenase promoter activity, providing the firstlink between this enzyme's activity and an established tumor-suppressorgene (19). Also of interest, over-expression of 15-lipoxygenase enhancesendogenous LXA₄ formation that in turn inhibits progression ofglomerulonephritis (20). Both ATL and LXA₄ share sites of action thatare receptor cell type, and tissue-specific, and together with theirstable analogs display potent anti-inflammatory actions (21, 23 and 14).

Endothelial cell migration is an essential component of the angiogenicprocess, providing directionality for the budding capillary toward theangiogenic stimulus (3). Therefore, endothelial migration was assessedwith ATL. VEGF (3 ng/ml) was added to the lower compartment of achemotaxis chamber and cell migration across a 10-μm pore-sizegelatin-coated filter was quantitated (FIG. 2). Results in FIG. 2Bshowed that ATL-1, LXA₄, 15-epi-LXA₄ and 15-R/S-methyl, LXA₄ (ATL-1shown as representative) gave concentration-dependent inhibition ofVEGF-stimulated HUVEC migration with a maximum level of inhibition(˜45%) at 10 nM ATL. As observed with proliferation assays, ATL-1, LXA₄,15-epi-LXA₄ or 15-R/S-methyl, LXA₄ alone, even at higher concentrations(100 nM), did not induce endothelial cell migration (FIG. 2A), findingswhich suggest that ATL, LXA₄, 15-epi-LXA₄ or 15-R/S-methyl, LXA₄ play arole in blocking the early stages of cell migration to sites ofneovascularization.

Like VEGF (FIG. 1), LTD₄ also stimulated proliferation of HUVEC(42±1.2%) with a maximum at 10 nM, similar to the response obtained withVEGF (52.7±1.6%)). In contrast, LTB₄ at 10 nM did not give a significantresponse with these cells (FIG. 3B). The mitogenic action of LTD₄ (10nM) was antagonized by exposure of the cells to ATL-1 (0.1-100 nM) withan IC₅₀ of ˜3 nM (FIG. 3A). LTD₄ did not enhance or inhibit theVEGF-stimulated proliferation.

The antiproliferative actions of native lipoxins were first found withthe human lung adenocarcinoma cell line (23) and recently with humanrenal mesangial cells (24). The present findings, together withendothelial cell results, draw attention to the potential regulatoryrole for endogenous ATL in proliferative diseases. The actions of LX,ATL and stable analogs are transduced by a high affinity transmembranereceptor (ALXR) identified in several cell types (for a review, seeChiang et al. (25)). In mesangial cells, LXA₄ interacts with its ownhigh affinity receptor (i.e., ALXR) as well as with a subclass ofpeptido-leukotriene receptors (cysLT₁), where LXA₄ is a partial agonist(24). In this regard, LXA₄ and its bioactive stable analogs effectivelydisplace [³H]LTD₄ specific binding to vascular endothelial cells (26).Also, recent findings provide the first evidence that ATL specificallyantagonizes LTD₄ specific binding at recombinant human cysLT₁ clonedfrom endothelial cells, as well as acts at specific LXA₄ receptors (21).Since ATL-1 proved to be a potent inhibitor of HUVEC proliferation(FIGS. 1, 2 and 3), it was determined whether ATL affected angiogenesisin vivo.

During chronic inflammation, new vessels are required not only for themaintenance of tissue perfusion, but also to allow increased cellulartraffic (27). Therefore, to this end angiogenesis was assessed in vivousing a well established murine chronic granulomatous air pouch model,and the 6-day time interval was selected because it was shown to givenear maximal vascular density (16). ATL-1 injected locally (10 μg/pouch)immediately before the administration of VEGF (1 μg/pouch) gave a ˜48%reduction in the vascular index (FIG. 4A). For comparison, ATL-1 (10μg/mouse or 0.4 mg/kg mouse) proved to be much more potent than otherdescribed anti-angiogenic agents that required much higher doses,including steroids at 1-3 mg/kg (16), or the COX-2 inhibitors, whichrequire 1-6 mg/kg (28).

FIG. 4B shows representative vascular casts from typical day 6 air pouchlinings. In the mice given VEGF (FIG. 4B, bottom left panel), there isan established neovasculature with an extremely high degree of vasculardensity compared to only slightly dilated capillaries in the ATL-treatedanimals, where there was routinely clearly reduced vascular density(FIG. 4B, bottom right panel). In another set of experiments,fluorescein isothiocyanate-dextran was used to visualize the vessels inthis region. In sharp contrast to the actions of ATL-1, when LTB₄,another lipoxygenase pathway product, was given alone at the same doseas ATL-1 (10 μg LTB₄/pouch), LTB₄ stimulated neovascularization (n=2).

FIG. 5 shows photomicrographs of the dorsal linings dissected at day 6.Again, profound angiogenesis was demonstrated with extensive vascularnetworks in VEGF-treated pouch (FIG. 5, bottom left panel). Here too,treatment with ATL-1 (10 μg/pouch) gave striking reduction ofVEGF-elicited vasculature, as exemplified by the lack of visible finecapillaries (FIG. 5, bottom right panel).

It is important to note that ATL-1 at this dose (10 μg/mouse i.v.) doesnot evoke apparent changes in mean arterial pressure, excluding apossible action of ATL-1 at the level of vascular tone. This isparticularly noteworthy because, at high doses, LXA₄ can stimulatevasodilation in certain vascular beds. The present in vivo experimentswere performed in separate series to evaluate histology and the presenceof a vascular marker by using immunohistochemical staining of the murineair pouch with the vascular endothelial cell marker CD31 (FIG. 6).

Platelet/endothelial cell adhesion molecule-1 (or CD31) is a member ofthe Ig superfamily that is strongly expressed at the endothelialcell-cell junction, is present on platelets as well as leukocytes, andis held to play a role in angiogenesis and in transendothelial migrationof leukocytes. Immunohistochemical staining for CD31 in the pouchesshowed that, in the VEGF-treated mice, strong specific endothelial cellstaining was present and identified a prominent vascular network (FIG.6C). In contrast, a marked diminution of vessels was observed inVEGF-treated mice that were also treated with the LXA₄ analog (FIG. 6D).The levels of mild nonspecific staining associated with these airpouches were essentially identical to those of the air pouch sectionsfrom mice treated with either vehicle alone (FIG. 6A) or with LX analogalone (FIG. 6B), namely, mild nonspecific staining of inflammatorycells, predominantly leukocytes and macrophages, that are known to beassociated with these air pouches created from murine skin. Takentogether, these findings indicate that ATL reduced VEGF-stimulatedangiogenesis in vivo, suggesting that LXA₄ and 15-epi-LXA₄ can regulatethese actions in vivo.

Results from many clinical and laboratory studies have demonstratedprotective effects of aspirin in several forms of human cancer,including lung, colon and breast cancer, yet its potential anti-cancermechanism is not clear (see Ref. 9). ASA is thought to act, in part, viareduction of angiogenesis, which might be related to the ability of ASAto inhibit prostanoid biosynthesis (7). More recently, ASA was found totrigger a novel switch in eicosanoid biosynthesis as the acetylation ofCOX-2 enables the enzyme to produce 15R-HETE that is converted to15-epi-lipoxins, also known as ATL, during cell-cell interactions invitro and in vivo (11, 12, 14). ATLs as well as their stable bioactiveanalogs are potent inhibitors of several key events in acuteinflammation, such as PMN chemotaxis and transmigration across bothendothelial and epithelial cells, as well as diapedesis frompostcapillary venules (13, 14). The analogs of ATL mimic both endogenousATL and LX actions and were designed to resist rapid enzymaticinactivation in vivo. Bioactive analogs of 15-epi-LXA₄ were also foundto complete at both the ALXR on leukocytes and the cysLT₁ receptorpresent on vascular endothelial cells. In addition, these novelaspirin-triggered mediators inhibit cytokine release and can act at thegene transcriptional level (29) to redirect local cytokine-chemokineaxis (30), actions that are both of interest in the angiogenic process(3). It should be noted that most if not all other eicosanoids examinedto date are pro-angiogenic including leukotrienes (e.g., LTD₄ and LTB₄,see the following table and FIG. 3) (5, 6, 28). In view of this, it wassurprising that ATL-1, LXA₄, 15-epi-LXA₄ and 15-R/S-methyl, LXA₄possessed antiangiogenic activity.

LXB₄-Induced HUVEC Proliferation Compound Percent of Proliferation14-epi-LXB₄ 0.1 nM 15.5 ± 1.0 14-epi-LXB₄ 1.0 nM 18.3 ± 1.6 14-epi-LXB₄10 nM 27.0 ± 5.0 14-epi-LXB₄ 100 nM 27.7 ± 1.3 15-epi-LXB₄ 0.1 nM 20.1 ±1.2 15-epi-LXB₄ 1.0 nM 26.1 ± 2.8 15-epi-LXB₄ 10 nM 34.0 ± 2.515-epi-LXB₄ 100 nM 35.2 ± 5.2 15-epi-LXB₄-acetylenic 0.1 nM 18.7 ± 1.615-epi-LXB₄-acetylenic 1.0 nM 23.7 ± 0.4 15-epi-LXB₄-acetylenic 10 nM36.4 ± 3.7 15-epi-LXB₄-acetylenic 100 nM 37.4 ± 5.3VEGF-Induced Proliferation: 50.4±4.0

In summary, the present results demonstrate that anaspirin-triggered-lipoxin, 15-epi-LXA₄ analog, is a potent inhibitor ofangiogenesis and of endothelial cell proliferation in vivo. Togetherthese results reveal a novel action of 15-epi-lipoxins and suggest arole for the aspirin-triggered lipoxin circuit (14) as a potentialmechanism that can contribute to aspirin's recognized anti-angiogenicand anti-inflammatory properties (2, 7, 10). With increasing insightinto the fundamental role of angiogenesis within a broad range ofphysiological as well as disease processes (1-3), the modulation ofvascular growth could be a previously unappreciated and importantstrategic action for ATL, the natural endogenous lipoxin mimetic andtheir synthetic analogs.

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A critical    assessment of the use of microculture tetrazolium assays to measure    cell growth and function. Growth Regulation 5:69-84.-   16. Colville-Nash, P. R., C. A. S. Alam, I. Appleton, J. R.    Brown, M. P. Seed, and D. A. Willoughby. 1995. The pharmacological    modulation of angiogenesis in chronic granulomatous inflammation. J.    Pharmacol. Exp. Ther. 274:1463-1472.-   17. Gupta, K., S. Kshirsagar, W. Li, L. Gui, S. Ramakrishnan, P.    Gupta, P. Y. Law, and R. P. Hebbel. 1999. VEGF prevents apoptosis of    human microvascular endothelial cells via opposing effects on    MAPK/ERK and SAPK/JNK signaling. Exp. Cell Res. 247:495-504.-   18. Eliceiri, B. P., and D. A. Cheresh. 1999. The role of av    integrins during angiogenesis: insights into potential mechanisms of    action and clinical development. J. Clin. Invest. 103:1227-1230.-   19. Kelavkar, U. P., and K. F. Badr. 1999. 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Nicola,    editors. Academic Press, London. 2219-2233.-   26. Takano, T., S. Fiore, J. F. Maddox, H. R. Brady, N. A. Petasis,    and C. N. Serhan. 1997. Aspirin-triggered 15-epi-lipoxin A₄ and LXA₄    stable analogs are potent inhibitors of acute inflammation: Evidence    for anti-inflammatory receptors. J. Exp. Med. 185:1693-1704.-   27. Cotran, R. S., V. Kumar, and T. Collins, editors. 1999. Robbins    Pathologic Basis of Disease. 6th ed. W.B. Saunders Co.,    Philadelphia.-   28. Masferrer, J. L., A. Koki, and K. Seibert. 1999. COX-2    inhibitors: a new class of antiangiogenic agents. Ann. N.Y. Acad.    Sci. 889:84-86.-   29. Gewirtz, A. T., B. McCormick, A. S. Neish, N. A. Petasis, K.    Gronert, C. N. Serhan, and J. L. Madara. 1998. Pathogen-induced    chemokine secretion from model intestinal epithelium is inhibited by    lipoxin A₄ analogs. J. Clin. Invest. 101:1860-1869.-   30. Hachicha, M., M. Pouliot, N. A. Petasis, and C. N. Serhan. 1999.    Lipoxin (LX)A₄ and aspirin-triggered 15-epi-LXA₄ inhibit tumor    necrosis factor 1a-initiated neutrophil responses and trafficking:    regulators of a cytokine-chemokine axis. J. Exp. Med. 189:1923-1929.

Those skilled in the art will know, or be able to ascertain, using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims. Allpublications and references cited herein, including those in thebackground section, are expressly incorporated herein by reference intheir entirety.

1. A method to prevent, reduce or inhibit angiogenesis comprising thestep of administering to a subject an effective amount of a formula:

wherein X is R₁, OR_(x), or SR₁; wherein R₁ is (i) a hydrogen atom; (ii)an alkyl of 1 to 8 carbons atoms, inclusive, which can be straight chainor branched; (iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive; (iv)an aralkyl of 7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl; (vii) a detectablelabel molecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN); wherein Q₃ isO, S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) a hydrogen atom; (b) an alkyl of 1 to 8 carbon atoms, inclusive,which can be straight chain or branched; (c) a cycloalkyl of 3 to 6carbon atoms, inclusive; (d) an alkenyl of 2 to 8 carbon atoms,inclusive, which can be straight chain or branched; or (e) R_(a) Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is alkylene of 0 to 6carbons atoms, inclusive, which can be straight chain or branched; andwherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which can bestraight chain or branched; wherein R₄ is (a) a hydrogen atom; (b) analkyl of 1 to 6 carbon atoms, inclusive, which can be straight chain orbranched; wherein Y₁ or Y₂ is —OH, methyl, or —SH and wherein the otheris (a) a hydrogen atom (b) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0 to 3;and each Z, independently, is a cyano, a nitro, or a halogen atom; (c)an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;or (d) an alkoxy of 1 to 4 carbon atoms, inclusive; or Y₁ and Y₂ takentogether are (a) ═NH; or (b) ═O; wherein R₅ is (a) an alkyl of 1 to 9carbon atoms which can be straight chain or branched; (b)—(CH₂)_(n)—R_(i) wherein n=0 to 4 and R_(i) is (i) a cycloalkyl of 3 to10 carbon atoms, inclusive; (ii) a phenyl; or (iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl; (c) R_(a)Q_(a)R_(b)wherein Q_(a) is O or S; wherein R_(a) is alkylene of 0 to 6 carbonsatoms, inclusive, which can be straight chain or branched; wherein R_(b)is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chainor branched; (d) —C(R_(iii))(R_(iv))—R_(i) wherein R_(iii) and R_(iv)are each, independently: (i) a hydrogen atom; (ii) CH_(a)Z_(b) wherea+b=3, a=0 to 3, b=0+3, and wherein each Z, independently, is a cyano, anitro, or a halogen atom; (e) a haloalkyl of 1 to 8 carbon atoms,inclusive, and 1 to 6 halogen atoms, inclusive, straight chain orbranched; and wherein R₆, if present, is (a) a hydrogen atom; (b) analkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched;(c) a halogen; and pharmaceutically acceptable salts, esters, amides,and prodrugs thereof, such that angiogenesis is prevented, reduced orinhibited in the subject.
 2. The method of claim 1, further comprising apharmaceutically acceptable carrier.
 3. A method to prevent, reduce orinhibit angiogenesis comprising the step of administering to a subjectan effective amount of a formula:

wherein R₁ is (i) a hydrogen atom; (ii) an alkyl of 1 to 8 carbonsatoms, inclusive, which can be straight chain or branched; (iii) acycloalkyl of 3 to 10 carbon atoms, inclusive; (iv) an aralkyl of 7 to12 carbon atoms; (v) phenyl; (vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl; (vii) a detectablelabel molecule; or (viii) an alkenyl of 2 to 8 carbon atoms, inclusive,straight chain or branched; wherein n=1 to 10, inclusive; wherein R₂,R_(3a), and R_(3b) are each independently: (a) a hydrogen atom; (b) analkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain orbranched; (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive; (d) analkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chainor branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a)is alkylene of 0 to 6 carbons atoms, inclusive, which can be straightchain or branched; and wherein R_(b) is alkyl of 0 to 8 carbon atoms,inclusive, which can be straight chain or branched; wherein Y₁ or Y₂ is—OH, methyl, hydrogen, or —SH and wherein the other is (a) a hydrogenatom; (b) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0 to 3, and wherein eachZ, independently, is a cyano, a nitro, or a halogen atom; (c) an alkylof 2 to 4 carbon atoms, inclusive, straight chain or branched; (d) analkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched; orY₁ and Y₂ taken together are (a) ═NH; or (b) ═O; wherein Y₃ or Y₄ is—OH, methyl, hydrogen, or —SH and wherein the other is (a) a hydrogenatom; (b) CH_(a)Z_(b) wherein a+b=3, a=0 to 3, b=0 to 3, and whereineach Z, independently, is a cyano, a nitro, or a halogen atom; (c) analkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; (d)an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched;or Y₃ and Y₄ taken together are (a) ═NH; or (b) ═O; wherein Y₅ or Y₆ is—OH, methyl, hydrogen, or —SH and wherein the other is (a) a hydrogenatom; (b) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0 to 3 wherein each Z,independently, is a cyano, a nitro, or a halogen atom; (c) an alkyl of 2to 4 carbon atoms, inclusive, straight chain or branched; (d) an alkoxyof 1 to 4 carbon atoms, inclusive, straight chain or branched; or Y₅ andY₆ taken together are (a) ═NH; or (b) ═O; wherein R₅ is (a) an alkyl of1 to 9 carbon atoms which can be straight chain or branched; (b)—(CH₂)_(n)—R_(i) wherein n=0 to 4 and R_(i) is (i) a cycloalkyl of 3 to10 carbon atoms, inclusive; (ii) phenyl; or (iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl; (c) —R_(a)Q_(a)R_(b)wherein Q_(a) is —O— or —S—; and wherein R_(a) is alkylene of 0 to 6carbons atoms, inclusive, which can be straight chain or branched;wherein R_(b) is either alkyl of 0 to 8 carbon atoms, inclusive, whichcan be straight chain or branched or substituted phenyl; (d)—C(R_(iii))(R_(iv))—R_(i) wherein R_(iii) and R_(iv) are eachindependently: (i) a hydrogen atom; or (ii) CH_(a)Z_(b) where a+b=3, a=0to 3, b=0+3, and wherein each Z, independently, is a cyano, a nitro, ora halogen atom; or (e) haloalkyl of 1 to 8 carbon atoms, inclusive, and1 to 6 halogen atoms, inclusive, straight chain or branched; andpharmaceutically acceptable salts, esters, amides, and prodrugs thereof,such that angiogenesis is prevented, reduced or inhibited in thesubject.
 4. The method of claim 3, further comprising a pharmaceuticallyacceptable carrier.
 5. A method to prevent, reduce or inhibitangiogenesis comprising the step of administering to a subject aneffective amount of a formula:

wherein R_(a) is (a) a hydrogen atom; or (b) alkyl of 1 to 8 carbonatoms; wherein R_(b) and R_(c), if present, are each independently: (a)a hydrogen atom; (b) a hydroxyl, or a thiol; (c) a methyl or ahalomethyl; (d) a halogen; (e) an alkoxy of 1 to 3 carbon atoms; whereinR_(d) and R_(e), if present, are each independently: (a) a hydrogenatom; (b) a hydroxyl, or thiol; (c) a methyl or halomethyl; (d) ahalogen; (e) an alkoxy of 1 to 3 carbon atoms; or (f) an alkyls orhaloalkyl of 2 to 4 carbon atoms, inclusive, which can be straight chainor branched wherein R_(x), if present, is (a) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl; (b) a substitutedphenoxy

wherein Z_(i) through Z_(v) are as defined above; or

wherein Z_(i) through Z_(v) are as defined above; and pharmaceuticallyacceptable salts, esters, amides, and prodrugs thereof, such thatangiogenesis is prevented, reduced or inhibited in the subject.
 6. Themethod of claim 5, further comprising a pharmaceutically acceptablecarrier.
 7. A method to prevent, reduce or inhibit angiogenesiscomprising the step of administering to a subject an effective amount ofa formula:

wherein R_(a) is (a) a hydrogen atom; or (b) an alkyl of 1 to 8 carbonatoms; wherein R_(b) and R_(c) are each independently: (a) a hydrogenatom; (b) a hydroxyl or a thiol; (c) a halomethyl; (d) a halogen; (e) analkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched; or(f) an alkoxy of 1 to 3 carbon atoms, inclusive; wherein R_(d) andR_(e), if present, are each independently: (a) a hydrogen atom; (b) ahydroxyl, or a thiol; (c) a methyl or a halomethyl; (d) a halogen; (e)an alkoxy of 1 to 3 carbon atoms, inclusive; or (f) an alkyl orhaloalkyl of 2 to 4 carbon atoms, inclusive, which can be straight chainor branched; wherein R₅, if present, is (a) an alkyl of 1 to 9 carbonatoms which can be straight chain or branched; (b) —(CH₂)_(n,) —R_(i)wherein n=0 to 4 and R_(i) is (i) a cycloalkyl of 3 to 10 carbon atoms,inclusive; (ii) phenyl; or (iii) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl; (c) R_(a)Q_(a)R_(b)wherein Q_(a) is —O— or —S—; wherein R_(a) is alkylene of 0 to 6 carbonsatoms, inclusive, which can be straight chain or branched; wherein R_(b)is either alkyl of 0 to 8 carbon atoms, inclusive, which can be straightchain or branched or substituted phenyl; (d) —C(R_(iii))(R_(iv))13 R_(i)wherein R_(iii) and R_(iv) are each, independently: (i) a hydrogen atom;or (ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3 wherein each Z,independently, is a cyano, a nitro, or a halogen atom; or (e) haloalkylof 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive,straight chain or branched; and pharmaceutically acceptable salts,esters, amides, and prodrugs thereof, such that angiogenesis isprevented, reduced or inhibited in the subject.
 8. The method of claim7, further comprising a pharmaceutically acceptable carrier.
 9. A methodto prevent, reduce or inhibit angiogenesis comprising the step ofadministering to a subject an effective amount of a formula:

wherein R_(a) is (a) a hydrogen atom; or (b) alkyl of 1 to 8 carbonatoms, inclusive, straight chain or branched; and wherein R_(b) andR_(c) are each, independently: (a) a hydrogen atom; (b) a hydroxyl or athiol; (c) a halomethyl; (d) a halogen; (e) an alkyl of 1 to 3 carbonatoms, inclusive, straight chain or branched; (f) an alkoxy of 1 to 3carbon atoms, inclusive; and pharmaceutically acceptable salts, esters,amides, and prodrugs thereof, such that angiogenesis is prevented,reduced or inhibited in the subject.
 10. The method of claim 9, furthercomprising a pharmaceutically acceptable carrier.
 11. A method toprevent, reduce or inhibit angiogenesis comprising the step ofadministering to a subject an effective amount of a formula:

wherein R_(a) is (i) a hydrogen atom; (ii) an alkyl of 1 to 8 carbonsatoms, inclusive, which can be straight chain or branched; or (iii) adetectable label molecule; wherein n=1 to 10, inclusive; wherein Y₂,R_(3a), and R_(3b) are each, independently: (a) a hydrogen atom; (b) analkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain orbranched; (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive; (d) analkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chainor branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a)is alkylene of 0 to 6 carbons atoms, inclusive, which can be straightchain or branched; and wherein R_(b) is alkyl of 0 to 8 carbon atoms,inclusive, which can be straight chain or branched; wherein Y₁ is —OH,methyl, or —SH; wherein Y₂ is (a) a hydrogen atom; (b) CH_(a)Z_(b) wherea+b=3, a=0 to 3, b=0 to 3 wherein each Z, independently, is a cyano, anitro, or a halogen atom; or (c) an alkyl of 2 to 4 carbon atoms,inclusive, straight chain or branched; wherein Y₃ and Y₅ are eachindependently: (a) a hydrogen atom; (b) CH_(a)Z_(b) wherein a+b=3, a=0to 3, b=0 to 3 and wherein each Z, independently, is a cyano, a nitro,or a halogen atom; or (c) an alkyl of 2 to 4 carbon atoms, inclusive,straight chain or branched; wherein Y₄ and Y₆ are each, independently(a) a hydrogen atom; (b) an alkyl of 2 to 4 carbon atoms, inclusive,straight chain or branched; (c) an alkoxy of 1 to 4 carbon atoms,inclusive, straight chain or branched; or (d) a hydroxyl or thiol; andwherein R₅ is (a) an alkyl of 1 to 9 carbon atoms which can be straightchain or branched; (b) —(CH₂)_(n)—R_(i) wherein n=0 to 3 and R_(i) is(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive; (ii) phenyl; (iii)substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl; (c) —R_(a)Q_(a)R_(b)wherein Q_(a) is —O— or —S—; wherein R_(a) is alkylene of 0 to 6 carbonsatoms, inclusive, which can be straight chain or branched; wherein R_(b)is (a) a substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichcan be a straight chain or branched, and hydroxyl; (b) a substitutedphenoxy

wherein Z_(i) through Z_(v) are as defined above; or

wherein Z_(i) through Z_(v) are as defined above; (d) a haloalkyl of 1to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive,straight chain or branched; and pharmaceutically acceptable salts,esters, amides, and prodrugs thereof, such that angiogenesis isprevented, reduced or inhibited in the subject.
 12. The method of claim11, further comprising a pharmaceutically acceptable carrier.
 13. Amethod to prevent, reduce or inhibit angiogenesis comprising the step ofadministering to a subject an effective amount of a formula:

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl; (vii) a detectablelabel molecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q_(4,) if present, are eachindependently O, S or NH; wherein one of R₂ and R₃, if present, is ahydrogen atom and the other is (a) H; (b) an alkyl of 1 to 8 carbonatoms, inclusive, which may be a straight chain or branched; (c) acycloalkyl of 3 to 6 carbon atoms, inclusive; (d) an alkenyl of 2 to 8carbon atoms, inclusive, which may be straight chain or branched; or (e)R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) is alkylene of 0 to6 carbons atoms, inclusive, which may be straight chain or branched andwherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched, provided when R_(b) is 0, then R_(b) is ahydrogen atom; wherein R₄ is (a) H; (b) an alkyl of 1 to 6 carbon atoms,inclusive, which may be a straight chain or branched; wherein R₅ is

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁, ifpresent, is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,inclusive, straight chain or branched, an alkoxy of 1 to 4 carbon atoms,inclusive, or CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0 to 3 and Z iscyano, nitro or a halogen; wherein R₆ is (a) H; (b) an alkyl from 1 to 4carbon atoms, inclusive, straight chain or branched; wherein T, ifpresent, is O or S; and pharmaceutically acceptable salts, esters,amides, and prodrugs thereof, such that angiogenesis is prevented,reduced or inhibited in the subject.
 14. The method of claim 13, furthercomprising a pharmaceutically acceptable carrier.
 15. A method toprevent, reduce or inhibit angiogenesis comprising the step ofadministering to a subject an effective amount of a formula:

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl; (vii) a detectablelabel molecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein R₄ is (a) H; (b) an alkyl of 1 to 6carbon atoms, inclusive, which may be a straight chain or branched; andpharmaceutically acceptable salts, esters, amides, and prodrugs thereof,such that angiogenesis is prevented, reduced or inhibited in thesubject.
 16. The method of claim 15, further comprising apharmaceutically acceptable carrier, wherein said pharmaceutical carrieris not a ketone.